Human monoclonal antibody human CD134 (OX40) and methods of making and using same

ABSTRACT

The invention provides antibodies that specifically bind to OX40 (CD134), referred to as OX40 antibodies, anti-OX40 or anti-OX40 antibodies. Invention antibodies that specifically bind to OX40 include mammalian (human, primate, etc.), humanized and chimeric anti-OX40 antibodies. Invention antibodies and antibody subsequences (fragments) that specifically bind to OX40 include purified and isolated antibodies, as well as pharmaceutical formulations thereof, are useful in various methods including treatment, screening and detection methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/087,436, filed Jun. 24, 2008, now U.S. Pat. No. 8,283,450, whichis a 371 of International Application No. PCT/US2006/045522, filed Nov.27, 2006, which claims the benefit of priority to U.S. ProvisionalApplication Nos. 60/739,659, filed Nov. 25, 2005, all of whichapplications are expressly incorporated herein by reference in theirentirety.

INTRODUCTION

The immune system is composed of multiple cell types that work toprotect the body from infectious diseases. This is dependent on therecognition of foreign antigens and the ability to distinguish self fromnon-self. In some cases the barrier between self and non-self is broken,leading to destruction of tissues by our own immune system. Theseautoimmune responses can lead to debilitating diseases such as diabetes,multiple sclerosis, and inflammatory bowel disease. One of the majormediators of these autoimmune responses is the T lymphocyte or T cell.Normally, T cells are tolerant to self-antigens, but in severalautoimmune disorders this tolerance is lost and the T cells mount immuneresponses to healthy tissues. Removal of the autoimmune T cells orblockade of their activation or survival ameliorates the diseasesymptoms. However, this generic depletion of T cells or prevention oftheir activation leads to immunosuppression where patients become highlysusceptible to infectious agents. New therapies are necessary that canspecifically target the effector T cells and reduce only theauto-reactive T cells.

SUMMARY

The invention provides isolated or purified antibodies that specificallybind to an epitope in an amino acid sequence of OX40 extracellulardomain (e.g., SEQ ID NO:50). Antibodies include human, humanized andchimeric antibodies that bind to mammalian (e.g., primate or human) OX40sequence (e.g., SEQ ID NO:49) Antibodies also include those that haveOX40 antagonist activity. Antibodies further include those that haveOX40 agonist activity.

In particular embodiments, an antibody has OX40 antagonist activity anddecreases or increases production of a cytokine or an interferon,survival or proliferation of activated, effector, memory or regulatory Tcells, expression of an anti-apoptotic or pro apoptotic protein. Inparticular non-limiting aspects, a cytokine is selected from IL-1, IL-2,IL-4, IL-5, IL-6, IL-9, IL-10, IL-14, IL-16, IL-17, IL-23, IL-26, TNF-α,interferon γ, and GM-CSF; and an anti-apoptotic or pro-apoptotic proteinis selected from Bcl-xL, Bcl-2, Bad and Bim.

In other particular embodiments, OX40 antibodies are produced by ahybridoma cell line denoted as 112F32 (ATCC No. PTA-7217, deposited Nov.17, 2005, 110801 University Blvd., Manassas, Va. 20110-2209), 112V8(ATCC No. PTA-7219, deposited Nov. 17, 2005, 110801 University Blvd.,Manassas, Va. 20110-2209), 112Y55 (ATCC No. PTA-7220, deposited on Nov.17, 2005, 110801 University Blvd., Manassas, Va. 20110-2209), 112Y131(ATCC No. PTA-7218, deposited on Nov. 17, 2005, 110801 University Blvd.,Manassas, Va. 20110-2209), and 112Z5 (ATCC No. PTA-7216, deposited onNov. 17, 2005, 110801 University Blvd., Manassas, Va. 20110-2209). Inadditional embodiments, OX40 antibodies bind to an amino acid sequenceto which the antibody produced by a hybridoma cell line denoted as112F32, 112V8, 112Y131, 112Y55, or 112Z5 binds; have an OX40 bindingaffinity within about 1-5000 fold of an antibody produced by a hybridomacell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5; havegreater or less OX40 binding affinity than an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5;have an OX40 binding affinity within about KD 10⁻⁶ M to about KD 10⁻¹³ Mof an antibody produced by a hybridoma cell line denoted as 112F32,112V8, 112Y131, 112Y55, or 112Z5; have the binding specificity of anantibody produced by a hybridoma cell line denoted as 112F32, 112V8,112Y131, 112Y55, or 112Z5; competes with an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5for binding to OX40; inhibits or prevents binding of an antibodyproduced by a hybridoma cell line denoted as 112F32, 112V8, 112Y131,112Y55, or 112Z5 to OX40, as determined in an ELISA assay (e.g.,inhibits at least 50% of the binding of an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5to OX40); or binds to the same epitope as that of 112V8, 112Y55 andY131, and inhibits or prevents OX40 binding to OX40 ligand (OX40L)(e.g., by 85% or more).

Antibodies additionally include those that specifically bind to OX40expressed on cells. In particular embodiments, the antibody specificallybinds OX40 expressed on non-T cells (e.g., natural killer cells,granulocytes, monocytes, B cells), or cell lines transfected with OX40(e.g., CHO cells, L929 cells or HELA cells). In a particular aspect, theantibody specifically binds to activated human T cells, not resting Tcells.

Antibodies include mature heavy or light chain variable regionsequences, for example, as shown in SEQ ID NO:7-10 and SEQ ID NO:44-49.Antibodies also include modified and variant forms such as substitutionswithin or outside of a constant region, a complementary determiningregion (CDR) or a framework (FR) region antibody of a mature heavy orlight chain variable region sequence, for example, as shown in SEQ IDNO:7-10 and SEQ ID NO:44-49. In particular aspects, substitutionsinclude conservative amino acid substitutions. In additional particularaspects, substitutions include amino acid substitutions of 1-3, 3-5,5-10 or more amino acid residues. In further particular aspects, anantibody has 80%-85%, 85%-90%, 90%-95%, 96%, 97%, 98%, 99%, or moreidentity to a sequence of mature heavy or light chain variable regionsequence as shown in SEQ ID NO:7-10 and SEQ ID NO:44-49.

Antibodies also include subsequences of mature heavy or light chainvariable region sequence, for example, as shown in SEQ ID NO:7-10 andSEQ ID NO:44-49. In particular aspects, a subsequence is selected fromFab, Fab′, F(ab′)₂, Fv, Fd, single-chain Fvs (scFv), disulfide-linkedFvs (sdFv) and V_(L) or V_(H).

Antibodies also include heterologous domains. In particular aspects, aheterologous domain includes a tag, detectable label or cytotoxic agent.

Modified and variant antibodies such as substitutions, subsequences andadditions can retain a detectable activity of an OX40 antibody as setforth herein. In one embodiment, a modified antibody retains adetectable activity of an antibody produced by a hybridoma cell linedenoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5. In anotherembodiment, a modified antibody modulates T cell expansion or survival,or numbers of activated, effector, memory or regulatory T cells, ordepleting activated, effector, memory or regulatory T cells. Inparticular aspects, a modified antibody inhibits OX40 signaling orreduces or deletes numbers of auto-reactive T cells specific for aself-antigen (e.g., a self antigen such as myelin basic protein, myelinoligodendrocyte glycoprotein, proteolipid protein, collagen, synovialjoint tissue antigens, insulin, glutamic acid decarboxylase, intestinalantigens, thyroid antigens, histone proteins, muscle antigens and skinantigens).

Antibodies also include those that compete with or do not compete withbinding of antibody L106 to OX40. In a particular embodiment, anantibody does not inhibit or prevent the binding of antibody L106 toOX40, as determined in an ELISA assay.

Antibodies further include those that affect a function or activity ofOX40. In particular embodiments, an antibody inhibits or prevents thebinding of OX40 ligand to OX40; inhibits or prevents the binding of OX40ligand to activated T cells; modulates OX40-mediated cell signaling(e.g., inhibits or prevents); modulates an OX40-mediated cell response;or OX40-mediated cell signaling (e.g., inhibits or prevents). Inparticular aspects, an OX40-mediated cell response comprises lymphocyteproliferation, cytokine expression, lymphocyte survival, activation ofNF-kB, maintenance of PKB (Akt) activity, or upregulation of survivin.In further embodiments, an antibody induces lysis of EL4-human OX40expressing cells or activated human T cells mediated by natural killercells, macrophages or neutrophils, for example, the percent (%) specificcell lysis induced at 10 μg/ml of antibody is about 15 to 75%, 25 to65%, or 30 to 60%, or 50-100%.

Antibodies further include those that have a function or activity invivo. In particular embodiments, an antibody reduces, decreases orprevents a symptom of graft versus host disease in an acute or chronicxenograft host disease model, or causes a remission or regression ofgraft versus host disease in an acute or chronic xenograft host diseasemodel (e.g., immunodeficient (SCID) mice that received human peripheralblood mononuclear cells (PBMC) after administration of anti-IL2Rbetachain antibody and a sublethal dose of irradiation). In particularaspects, a symptom of graft versus host disease is weight loss, hairloss, skin rash, hematuria, hydroperitoneum, and inflammatory cellinfiltrates in liver, intestinal tract, lung, skin, or death.

In additional embodiments, an antibody reduces, decreases or preventsinflammation in lung, skin, or bowel, or reduces, or decreases orprevents a symptom of an autoimmune disorder. In particular aspects, anantibody decreases or prevents a symptom of rheumatoid arthritis,multiple sclerosis, diabetes, Crohn's disease, inflammatory boweldisease, ulcerative colitis, celiac disease, psoriasis, proliferativelupus nephritis, granulomatous myopathy, or polymyositis.

Antibodies include monoclonal and polyclonal antibodies, any isotypes orsubclasses thereof. In particular aspects, the antibody is an IgG (e.g.,IgG1, IgG2, IgG3 or IgG4), IgA, IgM, IgE, or IgD isotype.

Antibodies can be included in pharmaceutical compositions In oneembodiment, an antibody includes a pharmaceutically acceptable carrieror excipient.

Antibodies can be encoded by nucleic acid sequences. In one embodiment,a nucleic acid encodes a sequence of mature heavy or light chainvariable region sequence, for example, as shown in SEQ ID NO:7-10 andSEQ ID NO:44-49, or a subsequence thereof. In particular aspects, thenucleic acid sequences include SEQ ID NO:3-6 and SEQ ID NO:38-43, orsequences degenerate with respect to SEQ ID NO:3-6 and SEQ ID NO:38-43.In additional particular aspects, a nucleic acid encodes an amino acidsequence having one or more substituted, added or deleted amino acidresidues of heavy or light chain variable region sequence as shown inSEQ ID NO:7-10 and SEQ ID NO:44-49. In further particular aspects, anucleic acid that encodes an amino acid sequence having one or moresubstituted, added or deleted amino acid residues of heavy or lightchain variable region sequence as shown in SEQ ID NO:7-10 and SEQ IDNO:44-49, has an activity of an antibody produced by a hybridoma cellline denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5 (e.g., hasbinding affinity for an epitope of OX40 extracellular domain). In stillfurther particular aspects, the nucleic acid sequence includes anexpression control sequence or a vector.

Antibodies can be produced by host cells and isolated cells. Inparticular embodiments, a host or isolated cell is a hybridoma cell orCHO cell. In an additional particular embodiment, an isolated cellexpresses an antibody having the binding specificity as antibodyproduced by a hybridoma cell line denoted as 112F32, 112V8, 112Y131,112Y55, or 112Z5.

The invention provides kits. In particular embodiments, a kit includesan antibody produced by a hybridoma cell line denoted as 112F32, 112V8,112Y131, 112Y55, or 112Z5, and instructions for administering theantibody to a subject in need of treatment with the antibody.

The invention provides in vivo methods, including treatment andtherapeutic methods. In particular embodiments, a method for treating achronic or acute immune disease or disorder, or a symptom of a chronicor acute immune disease or disorder in a subject in need of treatmentincludes administering to the subject an antibody or the pharmaceuticalcomposition effective to treat the chronic or acute immune disease ordisorder, or a symptom of the chronic or acute immune disease ordisorder in the subject. In particular aspects, treatment results inalleviating or ameliorating one or more adverse symptoms or physicalconsequences associated with a chronic or acute immune disease ordisorder.

In additional particular embodiments, a method for treating graft versushost disease includes administering to a subject in need of treatment anantibody or a pharmaceutical composition effective to treat graft versushost disease. In particular aspects, treatment reduces, decreases orprevents onset, frequency, duration or severity of one or more adversesymptoms or physical consequences associated with graft versus hostdisease (e.g., weight loss, hair loss, skin rash, hematuria,hydroperitoneum, inflammatory cell infiltrates in liver, intestinaltract, lung or death), results in a remission or regression of graftversus host disease, or results in preventing graft versus host disease.In additional aspects, a graft can include bone marrow, hematopoieticstem cells, peripheral blood stems cells or cord blood stem cells.

In further particular embodiments, a method for treating transplantrejection include administering to a subject in need of treatment anantibody or pharmaceutical composition effective to treat transplantrejection. In particular aspects, treatment reduces, decreases orprevents onset, frequency, duration or severity of one or more adversesymptoms or physical consequences associated with transplant rejection(e.g., an immune response against the transplant or transplant cell ortissue destruction), results in a remission or regression of transplantrejection, or results in preventing transplant rejection. In additionalaspects, a transplant can include kidney, heart, lung, skin, liver orpancreas cells, tissue or organ.

In still further particular embodiments, a method for reducing,decreasing or preventing inflammation includes administering to asubject in need of treatment an antibody or pharmaceutical compositioneffective to reduce, decrease, or prevent onset, frequency, duration orseverity of inflammation. In particular aspects, the inflammation ispresent in lung, joint, muscle, skin, central or peripheral nervoussystem, or bowel. In additional aspects, a treatment results in reducingonset, frequency, duration or severity of one or more adverse symptomsor physical consequences associated with inflammation.

In yet additional particular embodiments, a method for treating anautoimmune disorder includes administering to a subject in need oftreatment an antibody or pharmaceutical composition effective to reduce,decrease or prevent onset, frequency, duration or severity of a symptomof an autoimmune disorder. In particular aspects, the autoimmunedisorder includes rheumatoid arthritis, multiple sclerosis, diabetes,Crohn's disease, inflammatory bowel disease, ulcerative colitis, celiacdisease, psoriasis, proliferative lupus nephritis, granulomatousmyopathy, or polymyositis. In additional aspects, a treatment results inreducing, decreasing or preventing onset, frequency, duration orseverity of one or more adverse symptoms or physical consequencesassociated with an autoimmune disorder.

Additional methods that can result in treatment or therapy, whenpracticed in vivo, include modulating OX40 activity or function. Inparticular embodiments, a method for inhibiting or preventing anOX40-mediated cell response includes administering to a subject in needof inhibiting or preventing an OX40-mediated cell response an antibodyor pharmaceutical composition effective to inhibit or prevent anOX40-mediated cell response (e.g., lymphocyte proliferation, cytokineexpression, or lymphocyte survival). In further embodiments, a methodfor inhibiting or blocking binding of an OX40 ligand to activated Tcells includes administering to a subject in need of blocking,inhibiting or preventing binding of an OX40 ligand to activated T cellsan antibody or pharmaceutical composition effective to inhibit orprevent binding of an OX40 ligand to activated T cells. In additionalembodiments, a method for inhibiting or blocking binding of an OX40ligand to OX40 includes administering to a subject in need of blocking,inhibiting or preventing binding of an OX40 ligand to OX40 an antibodyor the pharmaceutical composition effective to inhibit or preventbinding of an OX40 ligand to OX40. In still further particularembodiments, a method for modulating OX40-mediated cell signalingincludes administering to a subject in need of modulating OX40-mediatedcell signaling an antibody or pharmaceutical composition effective tomodulate OX40-mediated cell signaling. In yet additional particularembodiments, a method for reducing numbers of activated, effector,memory or regulatory T cells includes administering to a subject in needof reduced numbers of activated, effector, memory or regulatory T cellsan amount of antibody sufficient to reduce numbers of activated,effector, memory or regulatory T cells.

In still additional particular embodiments, a method for decreasing thenumber of activated T cells in the blood, spleen, lymph nodes,intestines, liver, lung, or skin in an acute or chronic xenograft hostdisease model includes administering an amount of antibody to the acuteor chronic xenograft host disease model sufficient to decrease thenumber of activated T cells in the blood, spleen, lymph nodes,intestines, liver, lung, or skin.

In even additional particular embodiments, a method for treating adisease or disorder caused by activated, effector, memory or regulatoryT cells includes administering to a subject an amount of antibodysufficient to reduce, decrease or prevent progression of the disease ordisorder caused by activated, effector, memory or regulatory T cells, ordeplete activated, effector, memory or regulatory T cells. In particularaspects, the disease or disorder includes graft versus host disease,inflammation or an autoimmune disorder.

Subjects treatable in accordance with the invention include mammals(e.g., humans). In particular embodiments, a subject that is a candidatefor or has been treated for a chronic or acute immune disease ordisorder; a subject that is a candidate for or has been treated forgraft versus host disease; a subject that is a candidate for or has beentreated for transplant rejection; a subject that is a candidate for orhas been treated for inflammation; or a subject that is a candidate foror has been treated for an autoimmune disorder; a that is subject is acandidate for or has been treated for an OX40-mediated cell response.

Methods of the invention that include administration or delivery of OX40antibodies can be practiced by any acceptable method. In particularembodiments, an OX40 antibody is administered to a subject locally,regionally, or systemically.

The invention also provides methods for producing human OX40 antibodiesthat has OX40 antagonist activity. In one embodiment, a method includesadministering a human OX40 extracellular domain conjugated with human Fcrecombinant protein or activated human T cells to an animal capable ofexpressing human immunoglobulin (e.g., a transgenic mouse or transgeniccow); screening the animal for expression of human OX40 antibody;selecting an animal that produces a human OX40 antibody; isolating anantibody from the selected animal; and determining whether the humanOX40 antibody has OX40 antagonist activity.

The invention further provides methods for producing human OX40antibodies that inhibits or prevents OX40 binding to OX40 ligand(OX40L). In one embodiment, a method includes administering a human OX40extracellular domain conjugated with human Fc recombinant protein oractivated human T cells to an animal capable of expressing humanimmunoglobulin (e.g., a transgenic mouse or transgenic cow); screeningthe animal for expression of human OX40 antibody; selecting an animalthat produces a human OX40 antibody; isolating an antibody from theselected animal; and determining whether the human OX40 antibodyinhibits or prevents OX40 binding to OX40 ligand (OX40L).

The invention moreover provides non-human transgenic animals thatexpress an OX40 antibody. In various embodiment, the expressed OX40antibody is identical to an antibody produced by a hybridoma cell linedenoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5; binds to an epitopein an amino acid sequence of OX40 extracellular domain to which anantibody produced by a hybridoma cell line denoted as 112F32, 112V8,112Y131, 112Y55, or 112Z5; has an OX40 binding affinity within about1-5000 fold of an antibody produced by a hybridoma cell line denoted as112F32, 112V8, 112Y131, 112Y55, or 112Z5; has an OX40 binding affinitywithin about KD 10⁻⁶ M to about KD 10⁻¹² M of an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5;has the binding specificity of an antibody produced by a hybridoma cellline denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5; or competeswith an antibody produced by a hybridoma cell line denoted as 112F32,112V8, 112Y131, 112Y55, or 112Z5 for binding to OX40.

DRAWING DESCRIPTIONS

FIGS. 1A-F: Flow cytometric analysis with human anti-human OX40antibodies. A. Forward scatter versus side scatter profile of human CD4T cells after three days of stimulation with PHA and IL2. B-F. Activatedhuman T cells labeled with human anti-human OX40 antibodies or controls.The thick line represents staining on cells in the activated gate, whilethe thin line represents cells in the resting gate. Labeling of theactivated cells by the isotype control IgG antibodies is shown by thedotted line in the histograms. B. 112F32, C. 112V8, D. 112Y55, E.112Y131, and F. 112Z5.

FIG. 2: Staining of activated human T cells by human anti-human OX40monoclonal antibodies. The geometric mean fluorescence intensity datapresented are derived from an activated T cell gate similar to the onedepicted in FIG. 1A. These data were used to determine the KD and BMAXshown in Table 3.

FIGS. 3A-B: Five OX40 antibodies compete for binding to OX40. A. Percentinhibition of bound hOX40:mFc to coated antibody detected withanti-mouse IgG-HRP. B. Percent inhibition of bound hOX40:hFc to mouseantibody detected with sheep anti-human IgG-HRP. Negative numbers notshown.

FIGS. 4 A-B: Five OX40 antibodies compete for binding to OX40. A.Percent inhibition of activated T cells stained with blocking antibodiesand biotinylated anti-OX40 antibodies detected with SA-PE. B. Percentinhibition of activated human T cells labeled with mouse anti-human OX40antibodies and biotinylated anti-OX40 antibodies detected with SA-PE.Negative numbers not shown.

FIG. 5A-B: Blockade of OX40L binding to OX40 by human anti-human OX40monoclonal antibodies measured by ELISA and flow cytometry. A. Percentinhibition of bound ligand was detected with anti-FLAG-HRP secondaryantibody by ELISA. B. Percent inhibition of bound OX40L was detectedwith anti-FLAG-PE antibody by flow cytometry.

FIGS. 6A-C: Panels A-C represent three studies with three differentdonor pairs and depict the effect of anti-OX40 antibodies on T cellproliferation.

FIGS. 7A-C: Antibody 112V8G1 recombinant antibody ameliorated acutexenogenic graft versus host disease. A. Total gross pathology score. B.Single cell suspensions of the spleens analyzed by flow cytometry forthe presence of human T cells. The average number of T cells andstandard deviation of each population in each treatment group is shown.C. Human interferon gamma was measured in the serum of mice. The averageamount of interferon gamma in pg/ml is shown for each group of mice plusthe standard deviation.

FIGS. 8A-C: Antibody 112V8G1 ameliorates chronic xenogenic graft versushost disease. A. Total gross pathology scores of individual mice. B.Average number of human T cells in the spleens of mice plus standarddeviation as determined by flow cytometry. C. Average number of human Tcells in the peripheral lymph nodes of SCID mice at day forty-eight posttransfer of CD4 T cells.

FIGS. 9A-C: Antibody 112V8G4PE ameliorates acute xenogenic graft versushost disease when administered at day 0, 3, or 6. A. Total grosspathology scores of individual mice treated at day 0. B. Average numberof human T cells in the spleens at day 12 from mice treated at day 0plus standard deviation of the group. C. Total gross pathology scores ofindividual mice treated at day 3 or day 6.

FIG. 10: Percent specific lysis by anti-human OX40 human IgG1antibodies. Anti-human OX40 human IgG1 antibodies mediate ADCC ofEL4-human OX40 targets.

DETAILED DESCRIPTION

The invention is based at least in part on antibodies that specificallybind to OX40 (CD134), which, for example, can be referred to as OX40antibodies, anti-OX40 or anti-OX40 antibodies. Invention antibodies thatspecifically bind to OX40 include mammalian (human, primate, etc.),humanized and chimeric anti-OX40 antibodies. Invention antibodies andantibody subsequences (fragments) that specifically bind to OX40 includepurified and isolated antibodies, as well as pharmaceutical formulationsthereof.

OX40 is a 50 kilodalton (KDa) glycoprotein and a member of the tumornecrosis factor receptor superfamily (TNFRSF). The ligand for OX40,OX40L (also referred to as TNFSF4, CD252), has been reported to beexpressed on endothelial cells, activated antigen presenting cellsincluding macrophages, dendritic cells, B cells and natural killercells. Although not wishing to be bound by theory, binding between CD40on antigen presenting cells increases OX40L expression as canlipopolysaccharide (LPS). Expression of OX40 on T cells can be inducedfollowing signaling via the T cell antigen receptor. For example, OX40is expressed on recently activated T cells at the site of inflammation.CD4 and CD8 T cells can upregulate OX40 under inflammatory conditions.

OX40 is also referred to as CD134, TNFRSF4, ACT35 and TXGP1L. OX40includes mammalian (e.g., primate, human) forms of OX40. Invention OX40antibodies therefore include antibodies that specifically bind tomammalian OX40 sequences such as human OX40. OX40 sequences, such ashuman OX40 include polymorphic variants. One non-limiting example of afull length human OX40 is a sequence set forth as:

(SEQ ID NO: 49) MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI.

OX40 antibodies, anti-OX40 and anti-OX40 antibodies refer to anantibodies that specifically bind to OX40. Specific binding is thatwhich is selective for an epitope present in OX40. Specific binding canbe distinguished from non-specific binding using assays known in the art(e.g., immunoprecipitation, ELISA, flow cytometry, Western blotting).

OX 40 antibodies may bind to different proteins when all or a part of anantigenic epitope to which the antibody specifically binds is present ona different protein. Thus, depending on the extent of sequence orstructural homology of the OX40 epitope, an OX40 antibody mayspecifically bind to another protein that has high sequence orstructural homology to the OX40 epitope. Accordingly, OX40 antibodiesmay bind to different proteins when an epitope of sufficient sequence orstructural homology is present on a different protein.

Invention OX40 antibodies include isolated and purified antibodies.Antibodies of the invention, including isolated or purified OX40antibodies therefore do not include human beings.

The term “isolated” used as a modifier of a composition means that thecomposition is made by the hand of man or is separated from one or moreother components in a naturally occurring in vivo environment, typicallyby one or more manipulative steps or processes. Generally, compositionsso separated are substantially free of one or more materials with whichthey normally associate with in nature, for example, one or moreprotein, nucleic acid, lipid, carbohydrate, cell membrane. Thus, anisolated composition is separated from other biological components inthe cell of the organism in which the composition naturally occurs, orfrom the artificial medium in which it is produced (e.g., syntheticallyor through cell culture). For example, an isolated OX40 antibody can beobtained from an animal in which the antibody is produced (e.g., anon-transgenic mammal or a transgenic mammal, such as a rodent (mouse)or an ungulate (bovine) animal) and is separated from other polypeptidesand nucleic acid. Thus, serum containing the antibody obtained from theanimal is considered isolated. The term “isolated” does not excludealternative physical forms, for example, an isolated antibody couldinclude antibody subsequences, chimeras, multimers, or derivatizedforms.

The term “purified” used as a modifier of a composition refers to acomposition free of most or substantially all of the materials withwhich it typically associates with in nature. Purified antibodies aretypically removed from components normally present in the antibodymilieu. Thus, an antibody supernatant separated from antibody producinghybridoma cell culture is considered purified. Purified therefore doesnot require absolute purity and is context specific. Furthermore, a“purified” composition can be combined with one or more other molecules.Thus, the term “purified” does not exclude combinations of compositions.Purity can be determined by any appropriate method, including, forexample, UV spectroscopy, chromatography (e.g., HPLC, gas phase), gelelectrophoresis (e.g., silver or coomassie staining) and sequenceanalysis (peptide and nucleic acid).

“Purified” proteins and nucleic acid include proteins and nucleic acidsproduced by standard purification methods. The term also includesproteins and nucleic acid produced by recombinant expression in a hostcell as well as chemical synthesis. “Purified” can also refer to acomposition in which the level of contaminants is below a level that isacceptable to a regulatory agency for administration to a human ornon-human animal, for example, the Food and Drug administration (FDA).

Invention OX40 antibodies include antibodies that specifically bind toan epitope in an amino acid sequence of OX40 extracellular domain. Inparticular embodiments, exemplary OX40 antibodies specifically bind tothree “epitopes” on OX40, as determined by a cross-blocking assay. Anexemplary non-limiting human OX40 extracellular domain sequence is setforth as:

(SEQ ID NO: 50) MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSNDRCCHECRPGN GMVSRCSRSQ NTVCRPCGPG FYNDVVSSKPCKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYKPGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASNSSDAICEDRD PPATQPQETQ GPPARPITVQ PTEAWPRTSQ GPS.

Peptide epitopes typically are short amino acid sequences, e.g. aboutfive to 15 amino acids in length. Techniques for identifying epitopesare known in the art and are described, for example, in U.S. Pat. No.4,708,871. Briefly, a set of overlapping oligopeptides derived from OX40polypeptide may be synthesized and bound to a solid phase array of pins,with a unique oligopeptide on each pin. The array of pins may comprise a96-well microtiter plate, permitting one to assay 96 oligopeptidessimultaneously. Discontinuous epitopes may be identified similarly usinghighly overlapping peptide scans of different lengths (e.g., 6-mers to15-mers) immobilized at high density on a membrane support. Highantibody concentrations are used and binding is detected by indirectimmunodetection. If multiple binding sequences are identified and areseparated by intervening sequences but the individual peptides are notrecognized, then a discontinuous epitope has been identified. Theseparated sequences would be expected to form a continuous area on thesurface of the target protein and represent a conformational epitope(Reineke, et al. Protein Sci. 7:951 (1998). Alternatively, phage displaypeptide library kits (New England BioLabs) are commercially availablefor epitope mapping. These and other methods can be used to determinebinding affinity for every possible subset of consecutive amino acids inorder to identify the epitope that a particular antibody binds. Epitopesmay also be identified by inference when epitope length peptidesequences are used to immunize animals from which are obtainedantibodies that bind to the peptide sequence. Continuous epitopes canalso be predicted using computer programs such as BEPITOPE (Odorico etal., J. Mol. Recognit. 16:20 (2003)).

Antibodies of the invention are monoclonal or polyclonal immunoglobulinmolecules that belong to any antibody class such as IgM, IgG, IgA, IgE,IgD, and any subclass thereof. Exemplary subclasses for IgG are IgG₁,IgG₂, IgG₃ and IgG₄. A “monoclonal” antibody refers to an antibody thatis based upon, obtained from or derived from a single clone, includingany eukaryotic, prokaryotic, or phage clone. A “monoclonal” antibody istherefore defined structurally, and not the method by which it isproduced.

Particular exemplary antibodies that specifically bind to OX40 aredenoted as 112F32 (ATCC No. PTA-7217, deposited Nov. 17, 2005), 112V8(ATCC No. PTA-7219, deposited Nov. 17, 2005), 112Y55 (ATCC No. PTA-7220,deposited on Nov. 17, 2005), 112Y131 (ATCC No. PTA-7218, deposited onNov. 17, 2005), and 112Z5 (ATCC No. PTA-7216, deposited on Nov. 17,2005), which are human monoclonal anti-human OX40 antibodies (humanantibodies that bind to human OX40). Exemplary invention OX40 antibodies112F32, 112V8, 112Y55, 112Y131, and 112Z5 have mature heavy or lightchain variable region sequences as shown in SEQ ID NO:7-10 and SEQ IDNO:44-49. The designation of 112F32, 112V8, 112Y55, 112Y131, and 112Z5can refer to either the antibody or a cell line (e.g., hybridoma, CHOcell or other host cell) that produces the OX40 antibody.

Exemplary invention human anti-human OX40 antibodies were produced usingtrans-chromosomic mice (KM mice) (WO 02/43478, WO 02/092812, and Ishida,et al., IBC's 11^(th) Antibody Engineering Meeting. Abstract (2000))immunized with various forms of soluble recombinant human OX40(OX40-hIgG1), fusion protein (hOX40:hFc) or activated human T cells thatexpress OX40. Exemplary antibodies were identified that specificallylabeled activated human T cells and not resting T cells. Exemplaryantibodies detectably stained human OX40 stably transfected cell lines,EL4-OX40 and CHO-OX40, and not non-transformed parental cell lines,indicating that the antibodies specifically bind to human OX40.Exemplary antibodies also bind to rhesus macaque OX40 and cynomolgusmacaque OX40, but do not detectably bind to murine OX40.

Antibodies of the invention can have kappa or lambda light chainsequences, either full length as in naturally occurring antibodies,mixtures thereof (i.e., fusions of kappa and lambda chain sequences),and subsequences/fragments thereof. Naturally occurring antibodymolecules contain two kappa or two lambda light chains.

Invention OX40 antibodies also include, for example, antibodies thatspecifically bind to an amino acid sequence to which the antibodyproduced by a hybridoma cell line denoted as 112F32 (ATCC No. PTA-7217,deposited Nov. 17, 2005), 112V8 (ATCC No. PTA-7219, deposited Nov. 17,2005), 112Y131 (ATCC No. PTA-7218, deposited on Nov. 17, 2005), 112Y55(ATCC No. PTA-7220, deposited on Nov. 17, 2005), or 112Z5 (ATCC No.PTA-7216, deposited on Nov. 17, 2005) binds. Invention OX40 antibodiesfurther include, for example, antibodies that specifically bind to anOX40 extracellular domain to which an antibody produced by a hybridomacell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5 binds.Invention OX40 antibodies additionally include, for example, antibodiesthat have the binding specificity of an antibody produced by a hybridomacell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5. Such OX40antibodies typically exhibit partial or complete blocking, reduction orinhibition of 112F32, 112V8, 112Y131, 112Y55, or 112Z5 antibody bindingto OX40.

OX40 antibodies that specifically bind to an amino acid sequence towhich the antibody produced by a hybridoma cell line denoted as 112F32,112V8, 112Y131, 112Y55, or 112Z5 binds, and antibodies having thebinding specificity of 112F32, 112V8, 112Y131, 112Y55, or 112Z5 antibodycan be identified using competition binding assays. Antibodies can beselected based upon an ability to compete for binding of 112F32, 112V8,112Y131, 112Y55, or 112Z5 antibody to OX40. The ability of an antibodyto compete for binding of 112F32, 112V8, 112Y131, 112Y55, or 112Z5antibody to OX40, or to inhibit, reduce, decrease, prevent or blockbinding of 112F32, 112V8, 112Y131, 112Y55, or 112Z5 antibody to OX40,can be determined by various assays know in the art, including enzymelinked immunosorbent assay (ELISA). In particular aspects, an inventionantibody inhibits or prevents binding of an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5to OX40, as determined in an ELISA assay. In further aspects, aninvention antibody inhibits at least 50% of the binding of an antibodyproduced by a hybridoma cell line denoted as 112F32, 112V8, 112Y131,112Y55, or 112Z5 to OX40, as determined in an ELISA assay.

Invention OX40 antibodies also include antibodies that specifically bindOX40 and that do not prevent or block binding of mouse anti-human OX40antibody L106 (Becton Dickinson, catalog number 340420) to OX40. Furtherincluded are OX40 antibodies that specifically bind OX40 and that do notinhibit, reduce or decrease binding of antibody L106 to OX40. AntibodyL106 is described, for example, in U.S. Pat. No. 6,277,962, WO 95/12673and Schlossman et al., eds. (Leukocyte Typing V: White CellDifferentiation Antigens, Oxford: Oxford University Press (1995) pp1157-60)

Invention OX40 antibodies include antibodies that specifically bind toOX40 having greater or less affinity for OX40 than antibody produced bya hybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or112Z5. For example, antibodies having an OX40 binding affinity withinabout 1-10,000 fold (e.g., 2-5, 5-10, 10-100, 100-1000 or1000-10,000-fold greater or less affinity, or any numerical value orrange within or encompassing such values) of an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5are provided. Invention OX40 antibodies therefore also includeantibodies having greater or less OX40 binding affinity than antibodyproduced by a hybridoma cell line denoted as 112F32, 112V8, 112Y131,112Y55, or 112Z5. In particular embodiments, an invention OX40 antibodyhas an OX40 binding affinity within about KD 10⁻⁶ M to about KD 10⁻¹³ M,or any numerical value or range within or encompassing such values, ofan antibody produced by a hybridoma cell line denoted as 112F32, 112V8,112Y131, 112Y55, or 112Z5.

Binding affinity can be determined by association (K_(a)) anddissociation (K_(d)) rate. Equilibrium affinity constant, KD, is theratio of K_(a)/K_(d). Association (K_(a)) and dissociation (K_(d)) ratescan be measured using surface plasmon resonance (SPR) (Rich and Myszka,Curr. Opin. Biotechnol. 11:54 (2000); Englebienne, Analyst. 123:1599(1998)). Instrumentation and methods for real time detection andmonitoring of binding rates are known and are commercially available(BiaCore 2000, Biacore AB, Upsala, Sweden; and Malmqvist, Biochem. Soc.Trans. 27:335 (1999)). KD values can be defined as the OX40 antibodyconcentration required to saturate one half (50%) of the binding siteson OX40.

Invention OX40 antibodies include antibodies capable of binding to OX40present on one or more cells in vivo, in primary cell isolates, passagedcells, cultured cells and immortalized cells. Specific non-limiting celltypes that can express OX40 include activated and other T cells (e.g.,activated, effector, memory or regulatory T cells) and non-T cells.Examples of non-T cells include natural killer (NK) cells, granulocytes(neutrophils), monocytes and B cells. Cells that do not naturallyexpress OX40 can be made to express OX40, for example, by transfectingor transforming cells with an OX40 encoding nucleic acid. OX40antibodies capable of binding to OX40 can bind to one or moretransfected or transformed cells that express or produce OX40.

Invention antibodies include antibodies that bind to OX40 and modulatean OX40 function or activity in vivo or in vitro (e.g. in a subject). Asused herein, the term “modulate” and grammatical variations thereof,when used in reference to an OX40 activity or function, means that theOX40 activity or function is detectably affected, altered or changed.Thus, an OX40 antibody that modulates an OX40 activity or function is anantibody that detectably affects, alters or changes one or more OX40activities or functions, which can include, for example, binding of OX40to OX40 ligand, OX40 mediated signaling or an OX40-mediated orOX40-modulatable cell response, or another OX40 activity or function asset forth herein or otherwise known or knowable.

Various non-limiting OX40 activities and functions that can be modulatedinclude, for example, OX40-mediated signaling or an OX40-mediated orOX40-modulatable cell response, cell proliferation or expansion (e.g.,lymphocytes such as activated, effector, memory or regulatory T cells),cell survival or apoptosis (e.g., lymphocytes such as activated,effector, memory or regulatory T cells), cytokine (e.g., Th1, Th2 andnon Th1/Th2 cytokines) and interferon expression or production,anti-apoptotic or pro-apoptotic protein expression or production, andtreatment, prevention or amelioration of disorders, diseases,physiological conditions, pathologies and symptoms thereof. Specificcytokines modulated include but are not limited to IL-1, IL-2, IL-4,IL-5, IL-6, IL-9, IL-10, IL-14, IL-16, IL-17, IL-23, IL-26, TNF-α,interferon γ, and GM-CSF (in vivo or vitro). Specific anti-apoptotic orpro-apoptotic protein expression include but are not limited to Bcl-xL,Bcl-2, Bad and Bim. Other non-limiting activities or functions of OX40that can be modulated included, for example, activation of NF-kB,maintenance of PKB (Akt) activity, and upregulation of survivin(Ambrosini et al., Nat. Med. 3:917 (1997); and Song et al., Immunity22:621 (2005)).

Exemplary antibodies as set forth herein therefore include antibodiesthat modulate one or more OX40-mediated signaling or an OX40-mediated orinduced cell response, cell proliferation (e.g., activated, effector,memory or regulatory T cells), cell survival or apoptosis (e.g.,activated, effector, memory or regulatory T cells), cytokine (e.g., Th1,Th2 and other non Th1/Th2 cytokines, e.g., IL-17, IL-23 and IL-26) andinterferon expression or production such as Th1, Th2, non Th1/Th2, IL-1,IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-14, IL-16, IL-17, IL-23, IL-26,TNF-α, interferon γ, and GM-CSF (in vivo or vitro), anti-apoptotic orpro-apoptotic protein expression (e.g., Bcl-xL, Bcl-2, Bad or Bim), andtreatment, prevention or amelioration of disorders, diseases,pathologies and symptoms thereof. In particular aspects, inventionantibodies modulate T cell expansion or survival, modulate numbers ofactivated, effector, memory or regulatory T cells, or deplete activated,effector, memory or regulatory T cells. In further particular aspects,invention antibodies reduce or delete numbers of auto-reactive T cellsspecific for a self-antigen (e.g., myelin basic protein (MBP), myelinoligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), collagen,synovial joint tissue antigens, insulin, glutamic acid decarboxylase,intestinal antigens, thyroid antigens, histone proteins, muscle antigensor skin antigens).

The term “antagonist” and grammatical variations thereof used inreference to an OX40 antibody, means an OX40 antibody that reduces,decreases, inhibits, retards, prevents or blocks OX40 binding to OX40ligand, or reduces, decreases, inhibits, retards, prevents or blocks anOX40 activity or function. The term “agonist” and grammatical variationsthereof used in reference to an OX40 antibody, means an OX40 antibodythat stimulates, increases, enhances, promotes or induces OX40 bindingto OX40 ligand, or stimulates, increases, enhances, promotes or inducesan activity or function induced by OX40. Invention OX40 antibodiestherefore include antagonist and agonist antibodies.

Invention OX40 antagonist antibodies, block, reduce, decrease, inhibitor prevent one or more activities or functions of OX40 in vitro or invivo, for example. In particular embodiments, OX40 antibodies that canblock, reduce, decrease, inhibit or prevent OX40 ligand (OX40L) bindingto OX40 (CD134) in the soluble form or OX40 expressed on the surface ofactivated T cells are provided. In additional embodiments, an OX40antibody induces lysis of EL4-human OX40 expressing cells or activatedhuman T cells in the presence of lytic effector cells (e.g., naturalkiller cells, macrophages or neutrophils). In particular aspects, thepercent (%) specific cell lysis induced at 10 μg/ml of antibody isbetween about 15 to 75%, 25 to 65%, or 30 to 60%, and can be as high as100%, depending upon background levels in the studies. In addition,incubation of exemplary OX40 antibodies with human peripheral bloodmononuclear cells (PBMC) co-cultured with PBMC from an allogeneic donorreduced cell proliferation by inhibiting allo-reactive CD4 and/or CD8 Tcells.

Invention OX40 antibodies include modified forms, such as substitutions(e.g., amino acid substitutions), additions and deletions (e.g.,subsequences or fragments), which can be referred to as “variants.” Suchmodified antibody forms and variants retain at least partial function oractivity of a reference OX40 antibody, for example, an OX40 antibodydenoted as 112F32, 112V8, 112Y55, 112Y131, and 112Z5, such as binding toOX40, or modulating an activity or function of OX40 (e.g., OX40signaling). Thus, a modified OX40 antibody can retain at least partialOX40 binding or the ability to modulate one or more OX40 functions oractivities (e.g., signaling, a cell response, etc.), for example.

As used herein, the term “modify” and grammatical variations thereof,means that the composition deviates from a reference composition.Modified proteins, nucleic acids and other compositions may have greateror less activity than or a distinct function from a reference unmodifiedprotein, nucleic acid or other composition.

In particular aspects, invention modified antibodies retain one or moreof an ability to modulate T cell expansion or survival, modulate numbersof activated, effector, memory or regulatory T cells, or depleteactivated, effector, memory or regulatory T cells. In further particularaspects, invention modified antibodies retain one or more of an abilityto reduce or delete numbers of auto-reactive T cells specific for aself-antigen or B cells producing antibodies specific for a self-antigen(e.g., a myelin basic protein (MBP), myelin oligodendrocyte glycoprotein(MOG), proteolipid protein (PLP), collagen, synovial joint tissueantigens, insulin, glutamic acid decarboxylase, intestinal antigens,thyroid antigens, histone proteins, muscle antigens or skin antigens).

In various embodiments, the antibody mature heavy or light chainvariable region sequence as shown in SEQ ID NOs:7-10 and SEQ IDNOs:44-49 has one or more amino acid substitutions within or outside ofa constant region, a complementary determining region (CDR) or aframework (FR) region. In particular aspects, an amino acid substitutionis a conservative substitution within or outside of a constant region, acomplementary determining region (CDR) or a framework (FR) region. Infurther various embodiments, invention antibodies are at least 60%, 70%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical, or anynumerical value or range within or encompassing such percent values, toa heavy or light chain sequence of an antibody produced by a hybridomacell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5, SEQ IDNOs:7-10 or SEQ ID NOs:44-49. Typical numbers of substituted residuesinclude 1-3, 3-5, 5-10 amino acid residues, or any numerical value orrange within or encompassing such values, or more amino acid residues.

Such antibodies that include amino acid substitutions can be encoded bya nucleic acid. Consequently, nucleic acid sequences encoding antibodiesthat include amino acid substitutions are also provided.

The terms “identity” or “identical” mean that two or more referencedentities are the same. Thus, where two protein sequences (e.g., OX40antibodies) are identical, they have the same amino acid sequence, atleast within the referenced region or portion. An “area of identity”refers to a portion of two or more referenced entities that are thesame. Thus, where two protein sequences are identical over one or moresequence regions they share identity within that region. “Substantialidentity” means that a molecule is structurally or functionallyconserved such that it has or is predicted to have at least partialfunction or activity of one or more of the reference molecule functionsor activities, or relevant/corresponding region or portion of thereference molecule to which it shares identity. Thus, a polypeptide(e.g., OX40 antibody) with substantial identity has or is predicted tohave at least partial activity or function as the reference polypeptide(e.g., OX40 antibody). For example, in a particular embodiment, an OX40antibody having one or more modifications (e.g., amino acidsubstitutions, deletions or additions) that retains at least partialactivity or function of unmodified OX40 antibody is considered to havesubstantial identity to the reference OX40 antibody.

Due to variation between structurally and functionally related proteins,the amount of sequence identity required to retain a function oractivity depends upon the protein, the region and the function oractivity of that region. Although there can be as little as 30% aminoacid sequence identity for proteins to retain a given activity orfunction, typically there is more, e.g., 50%, 60%, 75%, 85%, 90%, 95%,96%, 97%, 98%, identity to a reference sequence. The extent of identitybetween two sequences can be ascertained using a computer program andmathematical algorithm known in the art. Such algorithms that calculatepercent sequence identity (homology) generally account for sequence gapsand mismatches over the comparison region. For example, a BLAST (e.g.,BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol.215:403 (1990), publicly available through NCBI) has exemplary searchparameters as follows: Mismatch-2; gap open 5; gap extension 2. Forpolypeptide sequence comparisons, a BLASTP algorithm is typically usedin combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequencecomparison programs are also used to quantitate the extent of identity(Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson,Methods Mol. Biol. 132:185 (2000); and Smith et al., J. Mol. Biol.147:195 (1981)). Programs for quantitating protein structural similarityusing Delaunay-based topological mapping have also been developed(Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

A “conservative substitution” is the replacement of one amino acid by abiologically, chemically or structurally similar residue. Biologicallysimilar means that the substitution does not destroy a biologicalactivity, e.g., OX40 binding activity. Structurally similar means thatthe amino acids have side chains with similar length, such as alanine,glycine and serine, or a similar size. Chemical similarity means thatthe residues have the same charge or are both hydrophilic orhydrophobic. Particular examples include the substitution of onehydrophobic residue, such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, serine for threonine, and the like.

Modified antibodies also include one or more D-amino acids substitutedfor L-amino acids (and mixtures thereof), structural and functionalanalogues, for example, peptidomimetics having synthetic or non-naturalamino acids or amino acid analogues and derivatized forms. Modificationsinclude cyclic structures such as an end-to-end amide bond between theamino and carboxy-terminus of the molecule or intra- or inter-moleculardisulfide bond.

Additional specific non-limiting examples of amino acid modificationsinclude OX40 subsequences and fragments. Exemplary OX40 subsequences andfragments comprise a portion of an OX40 sequence to which an exemplaryOX40 antibody of the invention binds. Exemplary OX40 subsequences andfragments also include an immunogenic portion, for example, a portion ofOX40 that includes a sequence to which an exemplary OX40 antibody of theinvention binds.

In accordance with the invention, there are provided OX40 antibody andnucleic acids encoding OX40 antibody subsequences or fragments thatretain, at least a part of, a function or activity of an unmodified or areference OX40 antibody. As used herein, the term “subsequence” or“fragment” means a portion of the full length molecule. A subsequence ofan OX40 antibody encoding an OX40 antibody has at least one fewer aminoacids than a full length OX40 (e.g., one or more internal or terminalamino acid deletions from either amino or carboxy-termini). Asubsequence of OX40 antibody has at least one fewer amino acid than afull length OX40 antibody. A nucleic acid subsequence has at least oneless nucleotide than a full length comparison nucleic acid sequence.Subsequences therefore can be any length up to the full length nativeOX40.

OX40 antibody subsequences and fragments of the invention include amature heavy or light chain variable region sequence as shown in SEQ IDNOs:7-10 and SEQ ID NOs:44-49. OX40 antibody subsequences and fragmentsof the invention also include Fab, Fab′ and F(ab′)₂, Fv, Fd,single-chain Fv (scFv), disulfide-linked Fvs (sdFv), V_(L) and V_(H)domain fragments.

OX40 antibody subsequences and fragments can have the binding affinityas full length antibody, the binding specificity as full lengthantibody, or one or more activities or functions of as a full lengthantibody, e.g., a function or activity of OX40 antagonist or agonistantibody. The terms “functional subsequence” and “functional fragment”when referring to an antibody means an antibody portion that retains atleast a part of one or more functions or activities as full lengthreference antibody, e.g., a function or activity of OX40 antibody. Forexample, an antibody subsequence or fragment that binds to OX40 or afragment of OX40 is considered a functional subsequence.

Antibody subsequences and fragments can be combined. For example, V_(L)or V_(H) subsequences can be joined by a linker sequence thereby forminga V_(L)-V_(H) chimera. A combination of single-chain Fvs (scFv)subsequences can be joined by a linker sequence thereby forming anscFv-scFv chimera. OX40 antibody subsequences and fragments, includesingle-chain antibodies or variable region(s) alone or in combinationwith all or a portion of other OX40 antibody subsequences.

Antibody subsequences and fragments can be prepared by proteolytichydrolysis of antibody, for example, by pepsin or papain digestion ofwhole antibodies. Antibody subsequences and fragments produced byenzymatic cleavage with pepsin provide a 5S fragment denoted F(ab′)₂.This fragment can be further cleaved using a thiol reducing agent toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and the Fcfragment directly (see, e.g., U.S. Pat. Nos. 4,036,945 and 4,331,647;and Edelman et al., Methods Enymol. 1:422 (1967)). Other methods ofcleaving antibodies, such as separation of heavy chains to formmonovalent light-heavy chain fragments, further cleavage of fragments,or other enzymatic or chemical may also be used.

Proteins and antibodies, as well as subsequences and fragments thereof,can be produced by genetic methodology. Techniques include expression ofall or a part of the gene encoding the protein or antibody into a hostcell such as Cos cells or E. coli. The recombinant host cells synthesizefull length or a subsequence, for example, an scFv (see, e.g., Whitlowet al., In: Methods: A Companion to Methods in Enzymology 2:97 (1991),Bird et al., Science 242:423 (1988); and U.S. Pat. No. 4,946,778).Single-chain Fvs and antibodies can be produced as described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods Enzymol.203:46 (1991); Shu et al., Proc. Natl. Acad. Sci. USA 90:7995 (1993);and Skerra et al., Science 240:1038 (1988).

Modified forms include derivatized sequences, for example, amino acidsin which free amino groups form amine hydrochlorides, p-toluene sulfonylgroups, carbobenzoxy groups; the free carboxy groups from salts, methyland ethyl esters; free hydroxl groups that form O-acyl or O-alkylderivatives, as well as naturally occurring amino acid derivatives, forexample, 4-hydroxyproline, for proline, 5-hydroxylysine for lysine,homoserine for serine, ornithine for lysine, etc. Modifications can beproduced using methods known in the art (e.g., PCR based site-directed,deletion and insertion mutagenesis, chemical modification andmutagenesis, cross-linking, etc.).

Modified forms of protein (e.g., antibody), nucleic acid, and othercompositions include additions and insertions. For example, an additioncan be the covalent or non-covalent attachment of any type of moleculeto a protein (e.g., antibody), nucleic acid or other composition.Typically additions and insertions confer a distinct function oractivity.

Additions and insertions include fusion (chimeric) polypeptide ornucleic acid sequences, which is a sequence having one or more moleculesnot normally present in a reference native (wild type) sequencecovalently attached to the sequence. A particular example is an aminoacid sequence of another protein (e.g., antibody) to produce amultifunctional protein (e.g., multispecific antibody).

Antibodies of the invention also include chimeras or fusions with one ormore additional domains covalently linked thereto to impart a distinctor complementary function or activity. Antibodies include chimeras orfusions in which two or more amino acid sequences are linked togetherthat do not naturally exist in nature.

In accordance with the invention, there are provided OX40 antibodies andnucleic acid encoding OX40 antibodies that include a heterologousdomain. Heterologous domains can be an amino acid addition or insertion,but are not restricted to amino acid residues. Thus, a heterologousdomain can consist of any of a variety of different types of small orlarge functional moieties. Such moieties include nucleic acid, peptide,carbohydrate, lipid or small organic compounds, such as a drug, metals(gold, silver), etc.

Particular non-limiting examples of heterologous domains include, forexample, tags, detectable labels and cytotoxic agents. Specific examplesof tags and detectable labels include T7-, His-, myc-, HA- andFLAG-tags; enzymes (horseradish peroxidase, urease, catalase, alkalinephosphatase, beta-galactosidase, chloramphenicol transferase); enzymesubstrates; ligands (e.g., biotin); receptors (avidin); radionuclides(e.g., C¹⁴, S³⁵, P³², P³³, H³, I¹²⁵ and I¹³¹); electron-dense reagents;energy transfer molecules; paramagnetic labels; fluorophores(fluorescein, rhodamine, phycoerthrin); chromophores; chemi-luminescent(imidazole, luciferase); and bio-luminescent agents. Specific examplesof cytotoxic agents include diptheria, toxin, cholera toxin and ricin.

Linker sequences may be inserted between the protein (e.g., antibody),nucleic acid, or other composition and the addition or insertion (e.g.,heterologous domain) so that the two entities maintain, at least inpart, a distinct function or activity. Linker sequences may have one ormore properties that include a flexible structure, an inability to forman ordered secondary structure or a hydrophobic or charged character,which could promote or interact with either domain Amino acids typicallyfound in flexible protein regions include glycine, asparagine andserine. Other near neutral amino acids, such as threonine and alanine,may also be used in the linker sequence. The length of the linkersequence may vary (see, e.g., U.S. Pat. No. 6,087,329). Linkers furtherinclude chemical cross-linking and conjugating agents, such assulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidylsuberate (DSS), disuccinimidyl glutarate (DSG) and disuccinimidyltartrate (DST).

Further examples of additions include glycosylation, fatty acids,lipids, acetylation, phosphorylation, amidation, formylation,ubiquitinatation, and derivatization by protecting or blocking groupsand any of numerous chemical modifications. Other permutations andpossibilities will be readily apparent to those of ordinary skill in theart, and are considered to be within the scope of the invention.

Such modified sequences can be made using recombinant DNA technology viacell expression or in vitro translation. Polypeptide and nucleic acidsequences can also be produced by chemical synthesis using methods knownin the art, for example, an automated peptide synthesis apparatus (see,e.g., Applied Biosystems, Foster City, Calif.).

OX40 protein suitable for generating antibodies can be produced by anyof a variety of standard protein purification or recombinant expressiontechniques. For example, an OX40 sequence can be produced by standardpeptide synthesis techniques, such as solid-phase synthesis. A portionof the protein may contain an amino acid sequence such as a T7 tag orpolyhistidine sequence to facilitate purification of expressed orsynthesized protein. The protein may be expressed in a cell andpurified. The protein may be expressed as a part of a larger protein(e.g., a fusion or chimera) by recombinant methods.

Methods of producing polyclonal and monoclonal antibodies are known inthe art. For example, OX40 or an immunogenic fragment thereof,optionally conjugated to a carrier such as keyhole limpet hemocyanin(KLH) or ovalbumin (e.g., BSA), or mixed with an adjuvant such asFreund's complete or incomplete adjuvant, and used to immunize ananimal. Using hybridoma technology, splenocytes from immunized animalsthat respond to OX40 can be isolated and fused with myeloma cells.Monoclonal antibodies produced by the hybridomas can be screened forreactivity with OX40 or an immunogenic fragment thereof.

Animals that may be immunized include primates, mice, rats, rabbits,goats, sheep, cattle, or guinea pigs. Initial and any optionalsubsequent immunization may be through intravenous, intraperitoneal,intramuscular, or subcutaneous routes. Additionally, to increase theimmune response, antigen can be coupled to another protein such asovalbumin or keyhole limpet hemocyanin (KLH), thyroglobulin and tetanustoxoid, or mixed with an adjuvant such as Freund's complete orincomplete adjuvant. Initial and any optional subsequent immunizationmay be through intraperitoneal, intramuscular, intraocular, orsubcutaneous routes. Subsequent immunizations may be at the same or atdifferent concentrations of OX40 preparation, and may be at regular orirregular intervals.

Animals include those genetically modified to include human gene loci,which can be used to produce human antibodies. Transgenic animals withone or more human immunoglobulin genes are described, for example, inU.S. Pat. No. 5,939,598, WO 02/43478, and WO 02/092812. Usingconventional hybridoma technology, splenocytes from immunized mice thatare high responders to the antigen can be isolated and fused withmyeloma cells. A monoclonal antibody can be obtained that binds to OX40.

Additional methods for producing human polyclonal antibodies and humanmonoclonal antibodies are described (see, e.g., Kuroiwa et al., Nat.Biotechnol. 20:889 (2002); WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598).

The term “human” when used in reference to an antibody, means that theamino acid sequence of the antibody is fully human, i.e., human heavyand human light chain variable and human constant regions. Thus, all ofthe amino acids are human or exist in a human antibody. An antibody thatis non-human may be made fully human by substituting the non-human aminoacid residues with amino acid residues that exist in a human antibody.Amino acid residues present in human antibodies, CDR region maps andhuman antibody consensus residues are known in the art (see, e.g.,Kabat, Sequences of Proteins of Immunological Interest, 4^(th) Ed. USDepartment of Health and Human Services. Public Health Service (1987);Chothia and Lesk (1987). A consensus sequence of human V_(H) subgroupIII, based on a survey of 22 known human V_(H) III sequences, and aconsensus sequence of human V_(L) kappa-chain subgroup I, based on asurvey of 30 known human kappa I sequences is described in Padlan Mol.Immunol. 31:169 (1994); and Padlan Mol. Immunol. 28:489 (1991). Humanantibodies therefore include antibodies in which one or more amino acidresidues have been substituted with one or more amino acids present inany other human antibody.

OX40 antibodies include humanized antibodies which can be produced usingtechniques known in the art including, for example, CDR-grafting (EP239,400; WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan,Molecular Immunol. 28:489 (1991); Studnicka et al., Protein Engineering7:805 (1994); Roguska. et al., Proc. Nat'l. Acad. Sci. USA 91:969(1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Human consensussequences (Padlan, Mol. Immunol. 31:169 (1994); and Padlan, Mol.Immunol. 28:489 (1991)) have previously used to produce humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992);and Presta et al., J. Immunol. 151:2623 (1993)).

The term “humanized” when used in reference to an antibody, means thatthe amino acid sequence of the antibody has non-human amino acidresidues (e.g., mouse, rat, goat, rabbit, etc.) of one or morecomplementarity determining regions (CDRs) that specifically bind to thedesired antigen in an acceptor human immunoglobulin molecule, and one ormore human amino acid residues in the Fv framework region (FR), whichare amino acid residues that flank the CDRs. Antibodies referred to as“primatized” are within the meaning of “humanized” except that theacceptor human immunoglobulin molecule and framework region amino acidresidues may be any primate amino acid residue (e.g., ape, gibbon,gorilla, chimpanzees orangutan, macaque), in addition to any humanresidue. Human FR residues of the immunoglobulin can be replaced withcorresponding non-human residues. Residues in the CDR or human frameworkregions can therefore be substituted with a corresponding residue fromthe non-human CDR or framework region donor antibody to alter, generallyto improve, antigen affinity or specificity, for example. A humanizedantibody may include residues, which are found neither in the humanantibody nor in the donor CDR or framework sequences. For example, a FRsubstitution at a particular position that is not found in a humanantibody or the donor non-human antibody may be predicted to improvebinding affinity or specificity human antibody at that position.Antibody framework and CDR substitutions based upon molecular modelingare well known in the art, e.g., by modeling of the interactions of theCDR and framework residues to identify framework residues important forantigen binding and sequence comparison to identify unusual frameworkresidues at particular positions (see, e.g., U.S. Pat. No. 5,585,089;and Riechmann et al., Nature 332:323 (1988)).

OX40 antibodies include chimeric antibodies. As used herein, the term“chimeric” and grammatical variations thereof, when used in reference toan antibody, means that the amino acid sequence of the antibody containsone or more portions that are derived from, obtained or isolated from,or based upon two or more different species. For example, a portion ofthe antibody may be human (e.g., a constant region) and another portionof the antibody may be non-human (e.g., a murine heavy or murine lightchain variable region). Thus, an example of a chimeric antibody is anantibody in which different portions of the antibody are of differentspecies origins. Unlike a humanized or primatized antibody, a chimericantibody can have the different species sequences in any region of theantibody.

Methods for producing chimeric antibodies are known in the art (e.g.,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., J. Immunol. Methods 125:191 (1989); and U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397). Chimeric antibodies inwhich a variable domain from an antibody of one species is substitutedfor the variable domain of another species are described, for example,in Munro, Nature 312:597 (1984); Neuberger et al., Nature 312:604(1984); Sharon et al., Nature 309:364 (1984); Morrison et al., Proc.Nat'l. Acad. Sci. USA 81:6851 (1984); Boulianne et al., Nature 312:643(1984); Capon et al., Nature 337:525 (1989); and Traunecker et al.,Nature 339:68 (1989).

OX40 antibodies can also be generated using hybridoma, recombinant, andphage display technologies, or a combination thereof (see U.S. Pat. Nos.4,902,614, 4,543,439, and 4,411,993; see, also Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), 1980, and Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. 1988).

Suitable techniques that additionally may be employed in antibodymethods include OX40-based affinity purification, non-denaturing gelpurification, HPLC or RP-HPLC, size exclusion, purification on protein Acolumn, or any combination of these techniques. OX40 antibody isotypecan be determined using an ELISA assay, for example, a human Ig can beidentified using mouse Ig-absorbed anti-human Ig.

OX40 suitable for generating antibodies can be produced by any of avariety of standard protein purification or recombinant expressiontechniques known in the art. Forms of OX40 suitable for generating animmune response include OX40 subsequences, such as an immunogenicfragment. Additional forms OX40 include OX40 expressing cells, OX40containing preparations or cell extracts or fractions, partiallypurified OX40.

In accordance with the invention, there are provided isolated orpurified cells that express OX40 antibodies, subsequences and fragmentsthereof, and nucleic acids encoding OX40 antibodies, subsequences andfragments of the invention. In one embodiment, an isolated cellexpresses an antibody having the amino acid sequences of heavy or lightchain variable region of an antibody denoted as 112F32, 112V8, 112Y131,112Y55, or 112Z5. In another embodiment, an isolated cell expresses anantibody having the binding specificity as antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5.In a further embodiment, an isolated cell expresses an antibody thatcompetes with antibody produced by a hybridoma cell line denoted as112F32, 112V8, 112Y131, 112Y55, or 112Z5 for the binding of OX40. In anadditional embodiment, an isolated cell expresses an antibody that hasgreater or less binding affinity for OX40 than antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5.In particular aspects, binding affinity for OX40 is within about 1-5000fold of antibody produced by a hybridoma cell line denoted as 112F32,112V8, 112Y131, 112Y55, or 112Z5. In additional particular aspects,binding affinity for OX40 is within about KD10⁻⁶ M to about KD 10⁻¹³ Mof an antibody produced by a hybridoma cell line denoted as 112F32,112V8, 112Y131, 112Y55, or 112Z5. Specific non-limiting example ofisolated or purified cells that express OX40 antibodies, subsequencesand fragments thereof, and nucleic acids encoding OX40 antibodies,subsequences and fragments of the invention include spleen cells,hybridoma cells and CHO cells. The isolated or purified cells may be aplurality or population of cells from a primary cell isolate (e.g.,splenocytes), a secondary or passaged cell isolate, or an established orimmortalized cell culture (hybridoma or CHO cells).

In accordance with the invention, further provided are methods ofproducing antibodies that specifically bind to OX40. In one embodiment,a method for producing an OX40 antibody includes administering a humanOX40, subsequence or fragment (e.g., an OX40 extracellular domain),optionally conjugated with human Fc recombinant protein, to an animalcapable of expressing human immunoglobulin (e.g., transgenic mouse ortransgenic cow), screening the animal for expression of human OX40antibody, and selecting an animal that produces a human OX40 antibody,isolating an antibody from the selected animal. In one aspect, themethod determines whether the human OX40 antibody has OX40 antagonist oragonist activity.

In accordance with the invention, additionally provided are methods ofproducing human OX40 antibodies that inhibit or prevent OX40 binding toOX40 ligand (OX40L). In one embodiment, a method for producing a humanOX40 antibody includes administering OX40, subsequence or fragment(e.g., an OX40 extracellular domain), optionally conjugated with humanFc recombinant protein to an animal capable of expressing humanimmunoglobulin (e.g., transgenic mouse or transgenic cow), screening theanimal for expression of human OX40 antibody, selecting an animal thatproduces a human OX40 antibody, and isolating an antibody from theselected animal that produces human OX40 antibody. In one aspect, themethod determines whether the human OX40 antibody inhibits or preventsOX40 binding to OX40 ligand (OX40L).

In accordance with the invention, there are provided non-humantransgenic animals that express an OX40 antibody having one or more ofthe following characteristics: a) is identical to an antibody producedby a hybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or112Z5; b) binds to an epitope in an amino acid sequence of OX40extracellular domain to which an antibody produced by a hybridoma cellline denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5; c) has an OX40binding affinity within about 1-5000 fold of an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5;d) has an OX40 binding affinity within about KD 10⁻⁶ M to about KD 10⁻¹²M of an antibody produced by a hybridoma cell line denoted as 112F32,112V8, 112Y131, 112Y55, or 112Z5; e) has the binding specificity of anantibody produced by a hybridoma cell line denoted as 112F32, 112V8,112Y131, 112Y55, or 112Z5; or f) competes with an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5for binding to OX40.

In accordance with the invention, there are also provided isolated orpurified nucleic acids encoding OX40 antibodies, subsequences andfragments thereof. In various embodiments a nucleic acid sequenceencodes a sequence of mature heavy or light chain variable regionsequence as shown in SEQ ID NOs:7-10 and SEQ ID NOs:44-49, or asubsequence thereof. In additional embodiments a nucleic acid sequencecomprises any of SEQ ID NOs:3-6 and SEQ ID NOs:38-43, and subsequencesthereof. In a further embodiment a nucleic acid sequence comprises asequence degenerate with respect to any of SEQ ID NOs:3-6 and SEQ IDNOs:38-43, and subsequences thereof.

Nucleic acids can be of various lengths. Lengths of nucleic acids thatencode invention OX40 antibodies or subsequences thereof typically rangefrom about 100 nucleotides to 600 nucleotides, or any numerical value orrange within or encompassing such lengths, 100 to 150, 150 to 200, 200to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500to 550, or about 550 to 600 nucleotides in length, or any numericalvalue or range or value within or encompassing such lengths. Lengths ofnucleic acids that specifically hybridize to nucleic acids encodinginvention OX40 antibodies or subsequences thereof typically range fromabout 10-20, 20-30, 30-50, 50-100, 100-150, 150-200, 200-250, 250-300,300-400, 400-500, 500-600 nucleotides, or any numerical value or rangewithin or encompassing such lengths.

The terms “nucleic acid” and “polynucleotide” refer to at least two ormore ribo- or deoxy-ribonucleic acid base pairs (nucleotides) that arelinked through a phosphoester bond or equivalent. Nucleic acids includepolynucleotides and polynucleosides. Nucleic acids include single,double or triplex, circular or linear, molecules. Exemplary nucleicacids include but are not limited to: RNA, DNA, cDNA, genomic nucleicacid, naturally occurring and non naturally occurring nucleic acid,e.g., synthetic nucleic acid. Short nucleic acids and polynucleotides(e.g., 10-20, 20-30, 30-50, 50-100 nucleotides) are commonly referred toas “oligonucleotides” or “probes” of single- or double-stranded DNA.

Nucleic acids are provided that encode an amino acid sequence having oneor more substituted, added or deleted amino acid residues of heavy orlight chain variable region sequence as shown in any of SEQ ID NOs:7-10and SEQ ID NOs:44-49. In one embodiment the substituted, added ordeleted heavy or light chain variable region sequence has bindingaffinity for an epitope of OX40 extracellular domain. In anotherembodiment the substituted, added or deleted heavy or light chainvariable region sequence has an activity of an antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5,for example, modulating a function or activity of OX40.

The invention provides nucleic acids that hybridize to a nucleic acidthat encodes all or a subsequence of an OX40 antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, or 112Z5,that is at least 80-90% complementary or homologous to the nucleic acidsequence that encodes all or a subsequence or fragment of antibodyproduced by a hybridoma cell line denoted as 112F32, 112V8, 112Y131,112Y55, or 112Z5. In one embodiment, the nucleic acid sequence has alength from about 10-20, 20-30, 30-50, 50-100, 100-150, 150-200,200-250, 250-300, 300-400, 400-500, 500-600, nucleotides, or anynumerical value or range within or encompassing such lengths. Inparticular aspects, the nucleic acid sequence hybridizes to a heavy orlight chain variable region sequence, or portion thereof (e.g., a CDR,an FR, etc.) of an antibody produced by a hybridoma cell line denoted as112F32, 112V8, 112Y131, 112Y55, or 112Z5.

The term “hybridize” and grammatical variations thereof refer to thebinding between nucleic acid sequences. Hybridizing sequences willgenerally have more than about 50% homology to a nucleic acid thatencodes an amino acid sequence of a reference (e.g., OX40 antibody)sequence. The hybridization region between hybridizing sequencestypically is at least about 12-15 nucleotides, 15-20 nucleotides, 20-30nucleotides, 30-50 nucleotides, 50-100 nucleotides, 100 to 200, 300 to400 nucleotides or more, or any numerical value or range within orencompassing such lengths.

Nucleic acid sequences further include nucleotide and nucleosidesubstitutions, additions and deletions, as well as derivatized forms andfusion/chimeric sequences (e.g., encoding OX40 antibody fused to aheterologous domain). For example, due to the degeneracy of the geneticcode, nucleic acids include sequences and subsequences degenerate withrespect to nucleic acids that encode antibody produced by a hybridomacell line denoted as 112F32, 112V8, 112Y131, 112Y55, and 112Z5. Otherexamples are nucleic acids complementary to a sequence that encodes anantibody produced by a hybridoma cell line denoted as 112F32, 112V8,112Y131, 112Y55, or 112Z5, subsequences and fragments thereof.

Nucleic acids can be produced using various standard cloning andchemical synthesis techniques. Techniques include, but are not limitedto nucleic acid amplification, e.g., polymerase chain reaction (PCR),with genomic DNA or cDNA targets using primers (e.g., a degenerateprimer mixture) capable of annealing to antibody encoding sequence.Nucleic acids can also be produced by chemical synthesis (e.g., solidphase phosphoramidite synthesis) or transcription from a gene. Thesequences produced can then be translated in vitro, or cloned into aplasmid and propagated and then expressed in a cell (e.g., a host cellsuch as yeast or bacteria, a eukaryote such as an animal or mammaliancell or in a plant).

In accordance with the invention, there are further provided nucleicacid sequences of the invention that include vectors. In one embodiment,a vector includes a nucleic acid sequence encoding an OX40 antibody,subsequence or fragment thereof. In particular embodiments, a vectorincludes a nucleic acid sequence encoding any antibody produced by ahybridoma cell line denoted as 112F32, 112V8, 112Y131, 112Y55, and112Z5, subsequence or fragment thereof.

Vectors are vehicles that can be manipulated by insertion orincorporation of a nucleic acid. Vectors include plasmid, viral,prokaryotic (bacterial) and eukaryotic (plant, fungal, mammalian)vectors. Vectors can be used for expression of nucleic acids in vitro orin vivo. Such vectors, referred to as “expression vectors,” are usefulfor introducing nucleic acids, including nucleic acids that encode OX40antibodies, subsequences and fragments thereof, and expressing theencoded protein in vitro (e.g., in solution or in solid phase), in cellsor in a subject in vivo.

Vectors can also be used for manipulation of nucleic acids. For geneticmanipulation “cloning vectors” can be employed, and to transcribe ortranslate the inserted nucleic acid, in vitro (e.g., in solution or insolid phase), in cells or in a subject in vivo.

A vector generally contains an origin of replication for propagation ina cell in vitro or in vivo. Control elements, including expressioncontrol elements, present within a vector, can be included to facilitatetranscription and translation, as appropriate.

Vectors can include a selection marker. A “selection marker” is a genethat allows for the selection of cells containing the gene. “Positiveselection” refers to a process of selecting cells that contain theselection marker upon exposure to the positive selection. Drugresistance is one example of a positive selection marker-cellscontaining the marker will survive in a culture medium containing thedrug, and cells lacking the marker will die. Selection markers includedrug resistance genes such as neo, which confers resistance to G418;hygr, which confers resistance to hygromycin; and puro, which confersresistance to puromycin. Other positive selection marker genes includegenes that allow identification or screening of cells containing themarker. These genes include genes for fluorescent proteins (GFP andGFP-like chromophores, luciferase), the lacZ gene, the alkalinephosphatase gene, and surface markers such as CD8, among others.“Negative selection” refers to a process in which cells containing anegative selection marker are killed upon exposure to an appropriatenegative selection agent. For example, cells which contain the herpessimplex virus-thymidine kinase (HSV-tk) gene (Wigler et al., Cell 11:223(1977)) are sensitive to the drug gancyclovir (GANC). Similarly, the gptgene renders cells sensitive to 6-thioxanthine.

Viral vectors include those based upon retroviral (lentivirus forinfecting dividing as well as non-dividing cells), foamy viruses (U.S.Pat. Nos. 5,624,820, 5,693,508, 5,665,577, 6,013,516 and 5,674,703;WO92/05266 and WO92/14829), adenovirus (U.S. Pat. Nos. 5,700,470,5,731,172 and 5,928,944), adeno-associated virus (AAV) (U.S. Pat. No.5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979),cytomegalovirus (CMV) based vectors (U.S. Pat. No. 5,561,063), reovirus,rotavirus genomes, simian virus 40 (SV40) or papilloma virus (Cone etal., Proc. Natl. Acad. Sci. USA 81:6349 (1984); Eukaryotic ViralVectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982; Sarver etal., Mol. Cell. Biol. 1:486 (1981); U.S. Pat. No. 5,719,054). Adenovirusefficiently infects slowly replicating and/or terminally differentiatedcells and can be used to target slowly replicating and/or terminallydifferentiated cells. Additional viral vectors useful for expressioninclude parvovirus, Norwalk virus, coronaviruses, paramyxo- andrhabdoviruses, togavirus (e.g., sindbis virus and semliki forest virus)and vesicular stomatitis virus (VSV).

Vectors including a nucleic acid can be expressed when the nucleic acidis operably linked to an expression control element. The term “operablylinked” refers to a physical or a functional relationship between theelements referred to that permit them to operate in their intendedfashion. Thus, a nucleic acid “operably linked” to an expression controlelement means that the control element modulates nucleic acidtranscription and as appropriate, translation of the transcript.

An “expression control element” or “expression control sequence” is apolynucleotide that influences expression of an operably linked nucleicacid. Promoters and enhancers are particular non-limiting examples ofexpression control elements and sequences. A “promoter” is a cis-actingDNA regulatory region capable of initiating transcription of adownstream (3′ direction) nucleic acid sequence. The promoter sequenceincludes nucleotides that facilitate transcription initiation. Enhancersalso regulate nucleic acid expression, but can function at a distancefrom the transcription start site of the nucleic acid to which it isoperably linked. Enhancers function when located at either 5′ or 3′ endsof the nucleic acid, as well as within the nucleic acid (e.g., intronsor coding sequences). Additional expression control elements includeleader sequences and fusion partner sequences, internal ribosome bindingsites (IRES) elements for the creation of multigene, or polycistronic,messages, splicing signal for introns, maintenance of the correctreading frame of the gene to permit in-frame translation of mRNA,polyadenylation signal to provide proper polyadenylation of thetranscript of interest, and stop codons.

Expression control elements include “constitutive” elements in whichtranscription of an operably linked nucleic acid occurs without thepresence of a signal or stimuli. Expression control elements that conferexpression in response to a signal or stimuli, which either increase ordecrease expression of operably linked nucleic acid, are “regulatable.”A regulatable element that increases expression of operably linkednucleic acid in response to a signal or stimuli is referred to as an“inducible element.” A regulatable element that decreases expression ofthe operably linked nucleic acid in response to a signal or stimuli isreferred to as a “repressible element” (i.e., the signal decreasesexpression; when the signal is removed or absent, expression increases).

For bacterial expression, constitutive promoters include T7, as well asinducible promoters such as pL of bacteriophage λ, plac, pap, ptac(ptrp-lac hybrid promoter). In insect cell systems, constitutive orinducible promoters (e.g., ecdysone) may be used. In yeast, constitutivepromoters include, for example, ADH or LEU2 and inducible promoters suchas GAL (see, e.g., Ausubel et al., In: Current Protocols in MolecularBiology, Vol. 2, Ch. 13, ed., Greene Publish. Assoc. & WileyInterscience, 1988; Grant et al., In: Methods in Enzymology, 153:516-544(1987), eds. Wu & Grossman, 1987, Acad. Press, N.Y.; Glover, DNACloning, Vol. II, Ch. 3, IRL Press, Wash., D.C., 1986; Bitter, In:Methods in Enzymology, 152:673-684 (1987), eds. Berger & Kimmel, Acad.Press, N.Y.; and, Strathern et al., The Molecular Biology of the YeastSaccharomyces eds. Cold Spring Harbor Press, Vols. I and II (1982)).

For mammalian expression, constitutive promoters of viral or otherorigins may be used. For example, CMV, SV40, or viral long terminalrepeats (LTRs) and the like, or inducible promoters derived from thegenome of mammalian cells (e.g., metallothionein IIA promoter; heatshock promoter, steroid/thyroid hormone/retinoic acid response elements)or from mammalian viruses (e.g., the adenovirus late promoter; mousemammary tumor virus LTR) are used.

Expression control elements include elements active in a particulartissue or cell type, referred to as “tissue-specific expression controlelements.” Tissue-specific expression control elements are typicallymore active in specific cell or tissue types because they are recognizedby transcriptional activator proteins, or other transcription regulatorsactive in the specific cell or tissue type, as compared to other cell ortissue types. Particular non-limiting examples of such expressioncontrol elements are promoters such as hexokinase II, COX-2,alpha-fetoprotein, carcinoembryonic antigen, DE3/MUC1, prostate specificantigen, C-erB2/neu, glucose-dependent insulinotropic polypeptide (GIP),telomerase reverse transcriptase and hypoxia-responsive promoter.

In accordance with the invention, host cells transformed or transfectedwith OX40 nucleic acid or vector of the invention are provided. Hostcells include but are not limited to prokaryotic and eukaryotic cellssuch as bacteria, fungi (yeast), plant, insect, and animal (e.g.,mammalian, including primate and human) cells. Non-limiting examples oftransformed cells include bacteria transformed with recombinantbacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acidexpression vectors; yeast transformed with recombinant yeast expressionvectors; plant cells infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); insect cells infected with recombinant virus expressionvectors (e.g., baculovirus); and animal cells infected with recombinantvirus expression vectors (e.g., retroviruses, adenovirus, vacciniavirus), or transformed animal cells engineered for stable expression. Anon-limiting example of a mammalian host cell that expresses OX40antibodies, subsequences and fragments thereof include a CHO cell. Thehost cells may be a plurality or population of cells from a primary cellisolate, a secondary or passaged cell isolated, or an established orimmortalized cell culture.

The term “transformed” or “transfected” when use in reference to a cell(e.g., a host cell) or organism, means a genetic change in a cellfollowing incorporation of an exogenous molecule, for example, a proteinor nucleic acid (e.g., a transgene) into the cell. Thus, a “transfected”or “transformed” cell is a cell into which, or a progeny thereof inwhich an exogenous molecule has been introduced by the hand of man, forexample, by recombinant DNA techniques.

The nucleic acid or protein can be stably or transiently transfected ortransformed (expressed) in the cell and progeny thereof. The cell(s) canbe propagated and the introduced protein expressed, or nucleic acidtranscribed. A progeny of a transfected or transformed cell may not beidentical to the parent cell, since there may be mutations that occurduring replication.

Typically, cell transfection or transformation employs a vector. Avector can be encompassed within a viral particle or vesicle andoptionally targeted to particular cell types by inclusion of a proteinon the particle or vesicle surface that binds to a target cell ligand orreceptor. Thus, the viral particle or vesicle itself, or a protein onthe viral surface can be made to target cells for transfection ortransformation in vitro, ex vivo or in vivo. Accordingly, viral andnon-viral vector means of delivery into cells, tissue or organs, invitro, in vivo and ex vivo are included.

Introduction of nucleic acid into target cells (e.g., host cells) canalso be carried out by methods known in the art such as osmotic shock(e.g., calcium phosphate), electroporation, microinjection, cell fusion,etc. Introduction of nucleic acid and polypeptide in vitro, ex vivo andin vivo can also be accomplished using other techniques. For example, apolymeric substance, such as polyesters, polyamine acids, hydrogel,polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers. A nucleic acid can be entrapped in microcapsules prepared bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly(methylmethacrolate) microcapsules, respectively, or in a colloidsystem. Colloidal dispersion systems include macromolecule complexes,nano-capsules, microspheres, beads, and lipid-based systems, includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.

Liposomes for introducing various compositions into cells are known inthe art and include, for example, phosphatidylcholine,phosphatidylserine, lipofectin and DOTAP (e.g., U.S. Pat. Nos.4,844,904, 5,000,959, 4,863,740, and 4,975,282; and GIBCO-BRL,Gaithersburg, Md.). Piperazine based amphilic cationic lipids useful forgene therapy also are known (see, e.g., U.S. Pat. No. 5,861,397).Cationic lipid systems also are known (see, e.g., U.S. Pat. No.5,459,127). Polymeric substances, microcapsules and colloidal dispersionsystems such as liposomes are collectively referred to herein as“vesicles.”

Invention OX40 antibodies are useful in treatment, therapeutic anddiagnostic applications, including clinical and diagnostic methods. Forexample, in a mouse model of acute and chronic graft versus host disease(GVHD) where human PBMC were transferred into irradiated severe combinedimmunodeficient (SCID) mice, human anti-human OX40 antagonist antibodiesreduced human T cell numbers and disease pathology when administeredbefore or after disease onset. Thus, invention OX40 antibodies arecapable of reducing, decreasing or preventing a symptom of graft versushost disease (GVHD) in an acute or chronic xenograft host disease model;and causing a remission or regression of graft versus host disease in anacute or chronic xenograft host disease model (e.g., an immunodeficient(SCID) mouse). Invention antibodies were also able to induce lysis ofOX40 expressing cells by natural killer cells. Although not wishing tobe bound by any theory, lysis of OX40 expressing cells may be due toantibody dependent cellular cytotoxicity (ADCC).

Invention OX40 antibodies are therefore useful in treatment or therapyof disorders and diseases which are amenable or may respond favorably bymodulating an OX40 activity or function. Thus, an OX40 antagonistantibody can be used to treat disorders, diseases, physiologicalconditions, pathologies and symptoms thereof that are amenable to or arelikely to respond favorably to reducing, decreasing, inhibiting,preventing or blocking OX40 activity or function, whereas an OX40agonist antibody can be used to treat disorders, diseases, physiologicalconditions, pathologies and symptoms thereof that are amenable to or arelikely to respond favorably to stimulating, increasing, enhancing,promoting or inducing OX40 activity or function.

In accordance with the invention, there are provided methods of treatingdisorders, diseases, conditions, pathologies and adverse symptoms orabnormalities associated with undesirable or aberrant immune responses.As used herein, an “undesirable immune response” or “aberrant immuneresponse” refers to any immune response, activity or function that isgreater or less than desired or physiologically normal. An undesirableimmune response, function or activity can be a normal response, functionor activity. Thus, normal immune responses so long as they areundesirable, even if not considered aberrant, are included within themeaning of these terms. An undesirable immune response, function oractivity can also be an abnormal response, function or activity. Anabnormal (aberrant) immune response, function or activity deviates fromnormal. Undesirable and aberrant immune responses can be humoral,cell-mediated or a combination thereof, either chronic or acute.

An example of an undesirable or aberrant immune response is an immuneresponse that is hyper-responsive, such as in the case of an autoimmunedisorder or disease (e.g., autoimmunity). Another example of anundesirable or aberrant immune response is where an immune responseleads to acute or chronic inflammation in any tissue or organ. Yetanother example of an undesirable or aberrant immune response is wherean immune response leads to destruction of cells, tissue or organ, suchas a transplant rejection, graft vs. host disease (GVHD), an autoimmunedisorder or disease, or inflammation. Still another example of anundesirable or aberrant immune response is where the immune response ishypo-responsive, such as where response to an antigen is less thandesired, e.g., tolerance has occurred.

Undesirable and aberrant immune responses therefore include chronic andacute immune disorders and diseases characterized by physiologicalconditions, pathologies and adverse symptoms or abnormalities, dependingupon the disorder or disease. Particular non-limiting examples of immunedisorders and diseases to which the invention applies include graftversus host disease (GVHD), transplant rejection, autoimmune disordersand inflammation.

In accordance with the invention, there are provided in vivo treatmentand therapeutic methods based, at least in part, upon the ability ofOX40 antibodies to modulate an OX40 activity or function. In particularmethods of treatment embodiments, disorders, diseases, physiologicalconditions, pathologies and symptoms amenable to or that may respond totreatment or therapy with invention OX40 antibodies include, forexample, a chronic or acute immune disease or disorder. In furthermethods of treatment embodiments, disorders, diseases, physiologicalconditions, pathologies and symptoms amenable to treatment or therapywith invention OX40 antibodies include, for example, inflammation,transplant rejection, graft versus host disease (GVHD), an autoimmunedisorder or disease, such as rheumatoid arthritis, multiple sclerosis,diabetes (e.g., insulin-dependent diabetes mellitus, IDDM, type Idiabetes), Crohn's disease (CD), inflammatory bowel disease (IBD),ulcerative colitis (UC), celiac disease, psoriasis, systemic lupuserythematosus (SLE), proliferative lupus nephritis, granulomatousmyopathy, polymyositis, and an OX40-mediated cell response that isundesirable or aberrant, for example.

The invention therefore provides methods in which OX40 antibodies,subsequences or fragments are used for reducing, decreasing, inhibiting,preventing and blocking a chronic or acute immune disease or disorder,inflammation, transplant rejection, graft versus host disease (GVHD), oran autoimmune disorder, such as rheumatoid arthritis, multiplesclerosis, diabetes (e.g., insulin-dependent diabetes mellitus, IDDM,type I diabetes), Crohn's disease (CD), inflammatory bowel disease(IBD), ulcerative colitis (UC), celiac disease, psoriasis, systemiclupus erythematosus (SLE), proliferative lupus nephritis, granulomatousmyopathy, polymyositis, or an OX40-mediated cell response that isundesirable or aberrant, treating and reducing, decreasing, inhibiting,preventing and blocking lung inflammation in airwayhyper-reactivity/hypersensitivity (e.g., asthma, allergic asthma) and inother tissues and organs. Specific non-limiting examples of a symptom ofgraft versus host disease treatable in accordance with the invention isweight loss, hair loss, skin rash, hematuria, hydroperitoneum, andinflammatory cell infiltrates in liver, intestinal tract, lung, skin,and death.

OX40 antibodies are also useful for reducing, decreasing, inhibiting,preventing and blocking inflammation present in lung, joint, muscle,skin, central or peripheral nervous system or bowel. OX40 antibodies areadditionally useful for reducing, decreasing, inhibiting, preventing andblocking inflammation that results from response of a subject toinfectious agents (e.g., bacterial, viral or parasitic infectiousagents).

Additional conditions amenable to treatment or therapy with OX40antibodies include, for example, osteoarthritis, psoriatic arthritis,encephalomyelitis, myasthenia gravis, autoimmune thyroiditis, atopicdermatitis, eczematous dermatitis, psoriasis, Sjögren's Syndrome,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, cutaneouslupus erythematosus, scleroderma, vaginitis, proctitis, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia (ITP), polychondritis, Wegener'sgranulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, interstitial lung fibrosis,Hashimoto's thyroiditis, autoimmune polyglandular syndrome,immune-mediated infertility, autoimmune Addison's disease, pemphigusvulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmunealopecia, Vitiligo, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, pernicious anemia, Guillain-Barre syndrome,Stiff-man syndrome, acute rheumatic fever, sympathetic ophthalmia,Goodpasture's syndrome, systemic necrotizing vasculitis,antiphospholipid syndrome, asthma (e.g., allergic asthma) and allergies.

As used herein, the terms “transplant,” “transplantation” andgrammatical variations thereof mean grafting, implanting, ortransplanting a cell, tissue or organ from one part of the body toanother part, or from one individual or animal to another individual oranimal. The transplanted cell, tissue or organ may therefore be anallograft or xenograft. Exemplary transplant cells include bone marrow,hematopoietic stem cells, peripheral blood stem cells or cord blood stemcells, allogeneic or non-allogeneic cells, and neural cells. Exemplarytransplant tissues include skin, blood vessel, eye and bone marrow.Exemplary transplant organs include kidney, heart, lung, pancreas andliver. The term also includes genetically modified cells, tissue andorgans, e.g., by ex vivo gene therapy in which the transformed cells,tissue and organs are obtained or derived from a subject (e.g., human oranimal) who then receives the transplant from a different subject (e.g.,human or animal).

Inflammation treatable in accordance with the invention includeinflammatory responses mediated by OX40 or amendable to treatment withOX40 antibody due to modulation of OX40, which in turn can modulate oneor more of cell proliferation, survival, death, or activity oflymphocytes (e.g., activated, effector, memory or regulatory T cells),etc. Methods (e.g., treatment) can result in a reduction in occurrence,frequency, severity, progression, or duration of inflammation. Exemplarysymptoms of inflammation include one or more of swelling, pain, rash,headache, fever, nausea, skeletal joint stiffness, or tissue or celldamage.

Inflammation may cause, directly or indirectly, cell, tissue or organdamage, either to multiple cells, tissues or organs, or to a single celltype, tissue type or organ. Exemplary tissues and organs that canexhibit damage include epidermal or mucosal tissue, gut, bowel,pancreas, thymus, liver, kidney, spleen, skin, or a skeletal joint(e.g., knee, ankle, hip, shoulder, wrist, finger, toe, or elbow).Treatment in accordance with the invention can result in reducing,inhibiting or preventing progression or worsening of tissue damage, orlead to regeneration of a damaged organ or tissue, e.g., skin, mucosum,liver.

As used herein, the terms “treat,” “treating,” “treatment” andgrammatical variations thereof mean a protocol, regimen, process orremedy performed on an individual subject or patient, in which it isdesired to obtain a physiologic effect or outcome in that patient.Methods of the invention therefore include, among other things,treatment and therapeutic methods that provide a measurable improvementor beneficial effect in a disorder, disease, physiological condition,pathology or a symptom of a given subject. A measurable improvement orbeneficial effect is any objective or subjective, transient, temporary,or long-term improvement in the disorder, disease, physiologicalcondition, pathology or symptom, or a reduction in onset, severity,duration or frequency of an adverse symptom associated with or caused bythe disorder, disease, physiological condition, pathology or condition.An invention method need not take effect immediately and there may besome delay, but over time eventual improvement or beneficial effect,stabilization or amelioration in a given subject will occur.

A satisfactory clinical endpoint of a treatment method in accordancewith the invention is achieved, for example, when there is anincremental or a partial decrease or reduction in severity, duration orfrequency of one or more pathologies, adverse symptoms or complications,associated with the disorder, disease, pathology or condition, orinhibition, reduction, prevention or reversal of one or more of thephysiological, pathological, biochemical or cellular manifestations orcharacteristics of the disorder, disease, physiological condition,pathology or symptom (e.g., a chronic or acute immune disease ordisorder, GVHD, transplant rejection, inflammation, or an autoimmunedisorder). A therapeutic benefit or improvement therefore can but neednot be a cure, or ablation of a majority or all pathologies, adversesymptoms or complications associated with or caused by the disorder,disease, physiological condition, pathology or symptom (e.g., a chronicor acute immune disease or disorder, GVHD, transplant rejection,inflammation, or an autoimmune disorder). Thus, a therapeutic benefit orimprovement need not result in a complete cure of any or allpathologies, adverse symptoms or complications associated with or causedby the disorder, disease, physiological condition, or pathology (e.g., achronic or acute immune disease or disorder, GVHD, transplant rejection,inflammation, or an autoimmune disorder). For example, partialreduction, decrease or inhibition, or a stabilization, or slowingprogression or worsening of a pathology, adverse symptom or complicationassociated with or caused by the disorder, disease, physiologicalcondition or pathology (e.g., a chronic or acute immune disease ordisorder, GVHD, transplant rejection, inflammation, or an autoimmunedisorder), even if only for a few days, weeks or months, or even if oneor more pathologies, adverse symptoms or complications associated withor caused by a the disease, disorder, pathology or condition remain(e.g., a chronic or acute immune disease or disorder, GVHD, transplantrejection, inflammation, or an autoimmune disorder) is a satisfactoryclinical outcome.

In various particular embodiments, treatment methods include alleviatingor ameliorating one or more adverse (physical) symptoms or consequencesassociated with a chronic or acute immune disorder or disease. Invarious additional particular embodiments, treatment methods includereducing, decreasing or preventing onset, frequency, duration orseverity of one or more adverse symptoms or physical consequencesassociated with graft versus host disease (e.g., weight loss, hair loss,skin rash, hematuria, hydroperitoneum, inflammatory cell infiltrates inliver, intestinal tract, lung and death), or result in a remission orregression of graft versus host disease, or result in preventing graftversus host disease. In various further particular embodiments,treatment methods include reducing, decreasing or preventing onset,frequency, duration or severity of one or more adverse symptoms orphysical consequences associated with transplant or graft rejection(e.g., an immune response against the transplant or graft, or transplantor graft cell destruction), or result in a remission or regression oftransplant or graft rejection, or result in preventing transplant orgraft rejection. In various still further particular embodiments,treatment methods include reducing, decreasing or preventing onset,frequency, duration or severity of one or more adverse symptoms orphysical consequences associated with rheumatoid arthritis, multiplesclerosis, diabetes (e.g., insulin-dependent diabetes mellitus, IDDM,type I diabetes), Crohn's disease (CD), inflammatory bowel disease(IBD), ulcerative colitis (UC), celiac disease, psoriasis, systemiclupus erythematosus (SLE), proliferative lupus nephritis, granulomatousmyopathy, polymyositis, or an OX40-mediated cell response that isundesirable or aberrant. In yet further various particular embodiments,treatment methods include reducing numbers or proliferation ofself-reactive cells or cells producing anti-self protein antibodies,inhibiting or preventing an increase in numbers, proliferation orsurvival of self-reactive cells or cells producing anti-self proteinantibodies.

In the case of an immune disorder or disease, a measurable improvementor beneficial effect includes modulating numbers, proliferation or anactivity of lymphocytes (e.g., activated, effector, memory or regulatoryT cells) towards physiologically normal baseline levels is considered asuccessful treatment outcome. An additional example of a measurableimprovement or beneficial effect for an immune disorder or immunedisease is an improvement in a histopathological change caused by orassociated with the immune disorder or disease. For example, preventingfurther or reducing skeletal joint infiltration or tissue destruction,or pancreas, thymus, kidney, liver, spleen, epidermal (skin) or mucosaltissue, gut or bowel infiltration or tissue destruction.

In accordance with the invention, there are provided methods ofblocking, inhibiting preventing, reducing, or decreasing binding of anOX40 ligand (OX40L) to activated T cells or OX40, in vitro or in vivo.In one embodiment, a method includes contacting activated T cells withan OX40 antibody effective to inhibit or prevent binding of an OX40ligand to activated T cells. In another embodiment, a method includescontacting OX40 with an OX40 antibody effective to inhibit or preventbinding of an OX40 ligand to OX40. In particular aspects, a method isperformed on a subject in need of blocking, reducing, decreasing,inhibiting or preventing binding of an OX40 ligand (OX40L) to activatedT cells, optionally with an OX40 pharmaceutical composition.

In accordance with the invention, there are provided methods ofmodulating OX40-mediated cell signaling, in vitro or in vivo. In oneembodiment, a method includes administering to a subject in need ofmodulating OX40-mediated cell signaling an OX40 antibody effective tomodulate OX40-mediated cell signaling.

In accordance with the invention, there are provided methods of reducingnumbers of activated, effector, memory or regulatory T cells, in vitroor in vivo. In one embodiment, a method includes administering to asubject in need of reduced numbers of activated, effector, memory orregulatory T cells an amount of OX40 antibody sufficient to reducenumbers of activated, effector, memory or regulatory T cells.

In accordance with the invention, there are provided methods of treatinga disease or disorder caused by activated, effector, memory orregulatory T cells. In one embodiment, a method includes administeringto a subject an amount of an OX40 antibody sufficient to reduce,decrease or prevent progression of the disease or disorder caused byactivated, effector, memory or regulatory T cells, or deplete activated,effector, memory or regulatory T cells. In particular aspects, thedisease or disorder comprises: graft versus host disease, inflammationor an autoimmune disorder.

In accordance with the invention, there are provided methods ofdecreasing the number of activated T cells in the blood, spleen, lymphnodes, intestines, liver, lung, or skin in a subject. In one embodiment,a method includes administering to the subject an amount of an OX40antibody sufficient to decrease the number of activated T cells in theblood, spleen, lymph nodes, intestines, liver, lung, or skin. In oneaspect, a method of decreasing the number of activated T cells in theblood, spleen, lymph nodes, intestines, liver, lung, or skin is in anacute or chronic xenograft host disease model.

Invention compositions and methods can be combined with any othertreatment or therapy that provides a desired effect. In particular,treatments, and therapies that have been characterized as having acomplementary or synergistic effect are applicable. Exemplary treatmentsand therapies include immune suppressive agents or drugs. Suchimmune-suppressive treatments and therapies can be performed prior to,substantially contemporaneously with any other methods of the invention,for example, a treatment or therapy.

The invention therefore provides combination methods in which themethods of the invention are used in a combination with any therapeuticregimen, treatment protocol or composition, such as an immunesuppressive protocol, agent or drug set forth herein or known in theart. In one embodiment, a method includes administering an OX40antibody, subsequence or fragment thereof and an immune suppressivetreatment, agent or drug. The immune suppressive treatment, agent ordrug can be administered prior to, substantially contemporaneously withor following administration of OX40 antibody, subsequence or fragmentthereof to a subject.

As used herein, the term “immune suppressive” or grammatical variationsthereof, when used in reference to a treatment, therapy, agent or drugmeans that the treatment, therapy, agent or drug provides an decrease,reduction, inhibition or prevention of an immune response, humoral orcell-mediated. Such therapies can suppress immune response generally orsystemically, or suppress immune response in a specific region orlocation.

Specific non-limiting classes of immune suppressive agents and drugsinclude alkylating agents, anti-metabolites, plant extracts, plantalkaloids, nitrosoureas, hormones (steroids such as glucocorticoids),nucleoside and nucleotide analogues. Specific examples of immunesuppressive drugs include cyclophosphamide, azathioprine, cyclosporin A,tacrolimus (FK506), rapamycin, methotrexate, FTY720, cox-2 inhibitorsand interleukins (e.g., IL-12).

Polyclonal and monoclonal antibodies are a particular example of animmune suppressive treatment or therapy. Immune suppressive antibodiesinclude, for example, infliximab (Remicade®), Rituxan®, Atgam®, andThymoglobuline®, Xenapax®, Simulect®, Humira®, Raptiva®, Tysabri®, andOrthoclone® (OKT3).

Methods of the invention also include, among other things, methods thatresult in a reduced need or use of another treatment protocol ortherapeutic regimen, process or remedy. For example, for inflammation,GVHD or an autoimmune disorder, a method of the invention has atherapeutic benefit if in a given subject a less frequent or reduceddose or elimination of an immune suppressive treatment or therapyresults.

Thus, in accordance with the invention, methods of reducing need or useof an immune suppressive treatment or therapy are provided. In oneembodiment, a method includes administering an OX40 antibody, fragmentor subsequent thereof to a subject that is undergoing or has undergonean immune suppressive therapy. In one aspect, a method includesadministering an OX40 antibody, fragment or subsequent thereof in anamount effective to reduce dosage, frequency or duration or eliminateneed for an immune-suppressive treatment or therapy of inflammation,GVHD or an autoimmune disorder. The methods can be performed prior to,substantially contemporaneously with or following administration of animmune-suppressive treatment or therapy.

The doses or “amount effective” or “amount sufficient” in a method oftreatment or therapy in which it is desired to achieve a therapeuticbenefit or improvement includes, for example, any objective orsubjective alleviation or amelioration of one, several or allpathologies, adverse symptoms or complications associated with or causedby the target disease, disorder, pathology or an adverse symptom orcomplication, to a measurable or detectable extent. Preventing,inhibiting or delaying a progression or worsening of the target disease,disorder, pathology or an adverse symptom or complication is also asatisfactory outcome. An amount sufficient or an amount effective meanssufficiency or effectiveness in a particular subject, not a group ofsubjects or the general population. Thus, the “amount effective” or“amount sufficient” will be enough to provide a therapeutic benefit to agiven subject.

An amount sufficient or an amount effective can but need not be providedin a single administration and, can but need not be, administered aloneor in combination with another treatment, protocol or therapeuticregimen. For example, the amount may be proportionally increased asindicated by the need of the subject, status of the disorder, disease orcondition treated or the side effects of treatment. In addition, anamount sufficient or an amount effective need not be sufficient oreffective if given in single or multiple doses without a secondtreatment, protocol or therapeutic regimen, since additional doses,amounts or duration above and beyond such doses, or additionaltreatments, protocols or therapeutic regimens may be included to beeffective or sufficient in a given subject. Amounts considered effectiveor sufficient also include amounts that result in a reduction of the useof another treatment, therapeutic regimen or protocol.

Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg/kgto about 100 mg/kg, and any numerical value or range or value withinsuch ranges. Greater or lesser amounts (doses) can be administered, forexample, 0.01-500 mg/kg, and any numerical value or range or valuewithin such ranges. Additional exemplary non-limiting amounts (doses)range from about 0.5-50 mg/kg, 1.0-25 mg/kg, 1.0-10 mg/kg, and anynumerical value or range or value within such ranges.

Methods of the invention may be practiced by any mode of administrationor delivery, or by any route, systemic, regional and localadministration or delivery. Exemplary administration and delivery routesinclude intravenous, intrarterial, intradermal, intramuscular,subcutaneous, intra-pleural, transdermal (topical), transmucosal,intra-cranial, intra-spinal, intra-ocular, rectal, oral (alimentary) andmucosal.

Methods of the invention may be practiced one or more times (e.g., 1-10,1-5 or 1-3 times) per day, week, month, or year. The skilled artisanwill know when it is appropriate to delay or discontinue administration.A non-limiting dosage schedule is 1-7 times per week, for 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or rangeor value within such ranges.

Of course, as is typical for any treatment or therapy, differentsubjects will exhibit different responses to treatment and some may notrespond or respond inadequately to a particular treatment protocol,regimen or process. Amounts effective or sufficient will thereforedepend at least in part upon the disease, disorder, pathology treated(e.g., inflammation, GVHD, transplant rejection, or an autoimmunedisorder, and if it is advanced, late or early stage), the therapeuticeffect desired, as well as the individual subject (e.g., thebioavailability within the subject, gender, age, etc.) and the subject'sresponse to the treatment or therapy which will be based, in part, upongenetic and epigenetic variability (e.g., pharmacogenomics).Furthermore, since every treated subject or patient may not respond to aparticular treatment or therapeutic method, protocol, regimen, processor remedy, the treatment or therapeutic methods are not required toachieve a particular measurable improvement or beneficial effect,clinical endpoint or desired outcome in each and every subject orpatient, or a given population so treated. Accordingly, a given subjector patient, or population may fail to respond, respond inadequately ormay exhibit undesirable responses, such as a side effect, to aninvention treatment or therapeutic method.

The terms “subject” and “patient” are used interchangeably herein andrefer to animals, typically mammals, such as humans, non-human primates(gorilla, chimpanzee, orangutan, macaque, gibbon), domestic animals (dogand cat), farm and ranch animals (horse, cow, goat, sheep, pig),laboratory and experimental animals (mouse, rat, rabbit, guinea pig).Subjects include disease model animals (e.g., such as mice, rats andnon-human primates) for studying in vivo efficacy (e.g., a GVHD animalmodel). Human subjects include children, for example, newborns, infants,toddlers and teens, between the ages of 1 and 5, 5 and 10 and 10 and 18years, adults between the ages of 18 and 60 years, and the elderly, forexample, between the ages of 60 and 65, 65 and 70 and 70 and 100 years.

Subjects include mammals (e.g., humans) in need of treatment, that is,they have a disease, disorder, pathology or symptom thereof that isamenable to or that may respond to treatment or therapy with an OX40antibody, subsequence or fragment. Subjects include those having or atrisk of having a chronic or acute immune disorder or disease,inflammation, GVHD, transplant rejection, inflammation, an autoimmunedisease or an OX40-mediated cell response. Subjects also include thosethat are candidates for or have been treated for one or more of: achronic or acute immune disorder or disease, GVHD, transplant rejection,inflammation, an autoimmune disease or an OX40-mediated cell response.Thus, a subject that is a candidate for or has received a cell, tissueor organ transplant or graft, such as a kidney, heart, lung, skin, eyeblood vessel, liver or pancreas transplant, or bone marrow,hematopoietic stem cell, peripheral blood stem cells or cord blood stemcells, either allogeneic or non-allogeneic cells, neural cells, is acandidate for treatment with an OX40 antibody.

Subjects further include those in need of an immune suppressivetreatment or therapy due to a lab or clinical diagnosis warranting suchtreatment, subjects undergoing an immune suppressive treatment ortherapy (e.g., due to a transplant), and subjects having undergone animmune suppressive treatment or therapy, and are at risk of relapse orrecurrence. At risk subjects include those with a family history of,genetic predisposition towards, or who have suffered previously from achronic or acute immune disease or disorder, inflammation, GVHD,transplant rejection, inflammation, an autoimmune disease or anOX40-mediated cell response. Such at risk subjects can be identifiedusing screens, such as auto-reactive T cells or anti-self proteinantibodies. For example, subjects that express rheumatoid factor are atrisk for rheumatoid arthritis. Subjects that express antibodies againstMBP, MOG or PLP, are at risk for multiple sclerosis.

At risk subjects can therefore be treated in order to inhibit or reducethe likelihood of developing a chronic or acute immune disease ordisorder, inflammation, GVHD, transplant rejection, inflammation, anautoimmune disease, or an OX40-mediated cell response, or suffering arelapse or recurrence of a chronic or acute immune disease or disorder,inflammation, GVHD, transplant rejection, inflammation, an autoimmunedisease, or an OX40-mediated cell response. The result of such treatmentcan be to reduce the risk of developing a chronic or acute immunedisease or disorder, inflammation, GVHD, transplant rejection,inflammation, an autoimmune disease, or an OX40-mediated cell response.

The antibodies, nucleic acids, and other compositions and methods of theinvention can be included in or employ pharmaceutical formulations. Suchpharmaceutical formulations are useful for treatment of, oradministration or delivery to, a subject in vivo locally, regionally orsystemically, or ex vivo.

Pharmaceutical formulations include “pharmaceutically acceptable” and“physiologically acceptable” carriers, diluents or excipients. The terms“pharmaceutically acceptable” and “physiologically acceptable” includesolvents (aqueous or non-aqueous), solutions, emulsions, dispersionmedia, coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration. Such formulations can becontained in a liquid; emulsion, suspension, syrup or elixir, or solidform; tablet (coated or uncoated, immediate, delayed, continuous, orpulsatile release), capsule (hard or soft, immediate, delayed,continuous, or pulsatile release), powder, granule, crystal, ormicrobead. Supplementary compounds (e.g., preservatives, antibacterial,antiviral and antifungal agents) can also be incorporated into theformulations.

Pharmaceutical formulations can be made to be compatible with aparticular local, regional or systemic administration or delivery route.Thus, pharmaceutical formulations include carriers, diluents, orexcipients suitable for administration by particular routes. Specificnon-limiting examples of routes of administration for compositions ofthe invention are parenteral, e.g., intravenous, intrarterial,intradermal, intramuscular, subcutaneous, intra-pleural, transdermal(topical), transmucosal, intra-cranial, intra-spinal, intra-ocular,rectal, oral (alimentary), mucosal administration, and any otherformulation suitable for the treatment method or administrationprotocol.

Solutions or suspensions used for parenteral application can include: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide.

Pharmaceutical formulations for injection include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Fluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Antibacterial andantifungal agents include, for example, parabens, chlorobutanol, phenol,ascorbic acid and thimerosal. Isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride can be includedin the composition. Including an agent which delays absorption, forexample, aluminum monostearate or gelatin can prolong absorption ofinjectable compositions.

Sterile injectable formulations can be prepared by incorporating theactive composition in the required amount in an appropriate solvent withone or a combination of above ingredients. Generally, dispersions areprepared by incorporating the active composition into a sterile vehiclecontaining a basic dispersion medium and any other ingredient. In thecase of sterile powders for the preparation of sterile injectablesolutions, methods of preparation include, for example, vacuum dryingand freeze-drying which yields a powder of the active ingredient plusany additional desired ingredient from a previously prepared solutionthereof.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays, inhalation devices (e.g., aspirators) orsuppositories. For transdermal administration, the active compounds areformulated into ointments, salves, gels, creams or patches.

The pharmaceutical formulations can be prepared with carriers thatprotect against rapid elimination from the body, such as a controlledrelease formulation or a time delay material such as glycerylmonostearate or glyceryl stearate. The formulations can also bedelivered using articles of manufacture such as implants andmicroencapsulated delivery systems to achieve local, regional orsystemic delivery or controlled or sustained release.

Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations are known to those skilled in the art. The materials canalso be obtained commercially from Alza Corporation (Palo Alto, Calif.).Liposomal suspensions (including liposomes targeted to cells or tissuesusing antibodies or viral coat proteins) can also be used aspharmaceutically acceptable carriers. These can be prepared according toknown methods, for example, as described in U.S. Pat. No. 4,522,811.

Additional pharmaceutical formulations appropriate for administrationare known in the art (see, e.g., Gennaro (ed.), Remington: The Scienceand Practice of Pharmacy, 20^(th) ed., Lippincott, Williams & Wilkins(2000); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7^(th) ed., Lippincott Williams & Wilkins Publishers (1999);Kibbe (ed.), Handbook of Pharmaceutical Excipients AmericanPharmaceutical Association, 3^(rd) ed. (2000); and Remington'sPharmaceutical Principles of Solid Dosage Forms, Technonic PublishingCo., Inc., Lancaster, Pa., (1993)).

The compositions used in accordance with the invention, including OX40antibodies, nucleic acids, treatments, therapies, agents, drugs andpharmaceutical formulations can be packaged in dosage unit form for easeof administration and uniformity of dosage. “Dosage unit form” as usedherein refers to physically discrete units suited as unitary dosagestreatment; each unit contains a quantity of the composition inassociation with the carrier, excipient, diluent, or vehicle calculatedto produce the desired treatment or therapeutic (e.g., beneficial)effect. The unit dosage forms will depend on a variety of factorsincluding, but not necessarily limited to, the particular compositionemployed, the effect to be achieved, and the pharmacodynamics andpharmacogenomics of the subject to be treated.

The invention further provides kits, including OX40 antibodies, nucleicacids, agents, drugs and pharmaceutical formulations, packaged intosuitable packaging material, optionally in combination with instructionsfor using the kit components, e.g., instructions for performing a methodof the invention. In one embodiment, a kit includes an OX40 antibody,subsequence or fragment and instructions for detecting OX40. In anotherembodiment, a kit includes an OX40 antibody, subsequence or fragment andinstructions for treating a subject in need of treatment (e.g., asubject having a disease, disorder, pathology, or condition amendable orthat may respond to treatment or therapy) with the OX40 antibody,subsequence or fragment. In one aspect, the instructions are fortreating a chronic or acute immune disease or disorder, inflammation,GVHD, transplant rejection, inflammation, an autoimmune disease or anOX40-mediated cell response. In further aspects, a kit includes animmune-suppressive treatment, therapy or agent.

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,etc.). The label or packaging insert can include appropriate writteninstructions, for example, a diagnostic or treatment method of theinvention. Instructions can therefore include instructions forpracticing any of the methods of the invention described herein. Thus,in various embodiments, a kit includes a label or packaging insertincluding instructions for practicing a method of the invention insolution, in vitro, in vivo, or ex vivo. In one aspect, the instructionsinclude administering or delivering the OX40 antibody, subsequence orfragment into a subject locally, regionally or systemically in atreatment or therapeutic method of the invention.

Instructions may additionally include indications of a satisfactoryclinical endpoint or any adverse symptoms or complications that mayoccur. Instructions may further include storage information, expirationdate, or any information required by regulatory agencies such as theFood and Drug Administration for use in a human subject.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin the kit, on a label affixed to the kit or packaging material, orattached to a vial or tube containing a component of the kit.Instructions may comprise audio or video medium and additionally beincluded on a computer readable medium, such as a disk (floppy disketteor hard disk), optical CD such as CD- or DVD-ROM/RAM, magnetic tape,electrical storage media such as RAM and ROM and hybrids of these suchas magnetic/optical storage media.

Invention kits can additionally include a buffering agent, apreservative, or a stabilizing agent. The kit can also include controlcomponents for assaying for activity, e.g., a control sample or astandard. Each component of the kit can be enclosed within an individualcontainer or in a mixture and all of the various containers can bewithin single or multiple packages.

In accordance with the invention further provided are cell-free (e.g.,in solution, in solid phase) and cell-based (e.g., in vitro or in vivo)methods of screening, detecting and identifying OX40. The methods can beperformed in solution, in vitro using a biological material or sample,and in vivo, for example, a sample of cells (e.g., lymphocytes) from ananimal. In one embodiment, a method includes contacting a biologicalmaterial or sample with an antibody that binds to OX40 under conditionsallowing binding of the antibody OX40; and assaying for binding of theantibody to OX40. The binding of the antibody to OX40 detects thepresence of OX40. In one aspect, OX40 is present on a cell or tissue. Inanother aspect, the biological material or sample is obtained from amammalian subject

The term “contacting,” when used in reference to a composition such as aprotein (e.g., OX40 antibody), material, sample, or treatment, means adirect or indirect interaction between the composition (e.g., OX40antibody) and the other referenced entity. A particular example ofdirect interaction is binding. A particular example of an indirectinteraction is where the composition acts upon an intermediary molecule,which in turn acts upon the referenced entity. Thus, for example,contacting a cell (e.g., a lymphocyte) with an OX40 antibody includesallowing the antibody to bind to the cell (e.g., through binding toOX40), or allowing the antibody to act upon an intermediary that in turnacts upon the cell.

The terms “assaying” and “measuring” and grammatical variations thereofare used interchangeably herein and refer to either qualitative orquantitative determinations, or both qualitative and quantitativedeterminations. When the terms are used in reference to binding, anymeans of assessing the relative amount, affinity or specificity ofbinding is contemplated, including the various methods set forth hereinand known in the art. For example, OX40 antibody binding to OX40 can beassayed or measured by an ELISA assay.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention relates. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed herein.

All publications, patents, Genbank accession numbers and otherreferences cited herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions,will control.

As used herein, singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “an antibody” includes a plurality of antibodiesand reference to “a treatment or therapy” can include multiple,sequential or simultaneous treatments or therapies, and so forth.

As used herein, all numerical values or numerical ranges include wholeintegers within or encompassing such ranges and fractions of the valuesor the integers within or encompassing ranges unless the context clearlyindicates otherwise. Thus, for example, reference to a range of 90-100%,includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%,91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%,etc., and so forth. In another example, reference to a range of 1-5,000fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc.,2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include, aspects that are not expressly included inthe invention are nevertheless disclosed.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

The Sequence Listing submitted herewith in computer readable form isincorporated herein by reference.

EXAMPLES Example 1

This example describes various materials and methods.

Antigen Preparation:

RNA was isolated from human peripheral blood mononuclear cells activatedfor two days with phytohemaglutinin (PHA, Remel, Lenexa, Kans.) usingTri Reagent (Invitrogen Corp., Carlsbad, Calif.). The sequence encodingthe extracellular domain of human OX40 was amplified byreverse-transcription polymerase chain reaction. The amplified productwas sequenced and confirmed to be identical to the published sequence ofhuman OX40 (WO95/12673, SEQ NO:1). The product was sub-cloned in frameinto the pfastbac-hFc baculovirus donor plasmid. This vector wasgenerated from the pfastbac plasmid (Invitrogen Corp.) and the Fcportion of human IgG1 (“hIgG1”). The hIgG1 sequence was excised from thepV11392.fc vector. Recombinant baculovirus were generated that encodedthe human OX40:hIgG1 fusion protein (hOX40:hFc). Trichoplusia niHigh-Five BTI-TN-5b1-4 (“Tn5”) insect cells (Invitrogen Corp.) wereinfected with the hOX40:hFc recombinant baculovirus for proteinproduction. The full-length OX40 sequence was amplified and cloned intothe pcDNA3.1 vector (Invitrogen Corp.) for expression in eucaryoticcells. EL-4 (ATCC TIB-39) and CHO-K1 (ATCC CCL-61) cells weretransfected using lipofectamine 2000 (Invitrogen Corp.) and stabletransfectants were selected using hygromycin B (Fisher Scientific,Pittsburgh, Pa.) or geneticin (Invitrogen Corp.), respectively.hOX40:hFc was conjugated to ovalbumin by glutaraldehyde coupling. One mgof hOX40:hFc was mixed with 0.5 mg of ovalbumin (Pierce, Rockford, Ill.)in 1 ml phosphate buffered saline (PBS). 50 μl of 1% EM gradegluataraldehyde (Sigma, St. Louis, Mo.) was slowly added and gentleshaking for five minutes mixed the solution. After three hours at roomtemperature 50 μl of 1 M ethanolamine (pH 7) was added and the solutionwas incubated for two more hours followed by buffer exchange on a NAP10column (Amersham Biosciences, Piscataway, N.J.) with phosphate bufferedsaline.

Nucleotide sequence of human OX40:human IgG1 fusion protein frominitiation codon (ATG) through human OX40 extracellular domain to end ofhuman Fc sequence (underlined) SEQ ID NO:1

ATGTGCGTGG GGGCTCGGCG GCTGGGCCGC GGGCCGTGTG CGGCTCTGCT CCTCCTGGGC 60CTGGGGCTGA GCACCGTGAC GGGGCTCCAC TGTGTCGGGG ACACCTACCC CAGCAACGAC 120CGGTGCTGCC ACGAGTGCAG GCCAGGCAAC GGGATGGTGA GCCGCTGCAG CCGCTCCCAG 180AACACGGTGT GCCGTCCGTG CGGGCCGGGC TTCTACAACG ACGTGGTCAG CTCCAAGCCG 240TGCAAGCCCT GCACGTGGTG TAACCTCAGA AGTGGGAGTG AGCGGAAGCA GCTGTGCACG 300GCCACACAGG ACACAGTCTG CCGCTGCCGG GCGGGCACCC AGCCCCTGGA CAGCTACAAG 360CCTGGAGTTG ACTGTGCCCC CTGCCCTCCA GGGCACTTCT CCCCAGGCGA CAACCAGGCC 420TGCAAGCCCT GGACCAACTG CACCTTGGCT GGGAAGCACA CCCTGCAGCC GGCCAGCAAT 480AGCTCGGACG CAATCTGTGA GGACAGGGAC CCCCCAGCCA CGCAGCCCCA GGAGACCCAG 540GGCCCCCCGG CCAGGCCCAT CACTGTCCAG CCCACTGAAG CCTGGCCCAG AACCTCACAG 600GGACCCTCCA GATCTTGTGA CAAAACTCAC ACATGCCCAC CGTGCCCAGC ACCTGAACTC 660CTGGGGGGAC CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT CATGATCTCC 720CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC ACGAAGACCC TGAGGTCAAG 780TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA AGACAAAGCC GCGGGAGGAG 840CAGTACAACA GCACGTACCG TGTGGTCAGC GTCCTCACCG TCCTGCACCA GGACTGGCTG 900AATGGCAAGG AGTACAAGTG CAAGGTCTCC AACAAAGCCC TCCCAGCCCC CATCGAGAAA 960ACCATCTCCA AAGCCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATCC 1020CGGGATGAGC TGACCAAGAA CCAGGTCAGC CTGACCTGCC TGGTCAAAGG CTTCTATCCC 1080AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG AGAACAACTA CAAGACCACG 1140CCTCCCGTGC TGGACTCCGA CGGCTCCTTC TTCCTCTACA GCAAGCTCAC CGTGGACAAG 1200AGCAGGTGGC AGCAGGGGAA CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC 1260CACTACACGC AGAAGAGCCT CTCCCTGTCT CCGGGTAAAT GA 1320

Amino acid sequence of human OX40-extracellular domain fused to the Fcportion of human IgG1 (underlined) SEQ ID NO:2

MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 60NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 120PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 180GPPARPITVQ PTEAWPRTSQ GPSRSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS 240RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL 300NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP 360SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN 420HYTQKSLSLS PGK 433Mice:

Human trans-chromosomic KM Mice™ (WO 02/43478, WO 02/092812, Ishida, etal., IBC's 11^(th) Antibody Engineering Meeting. Abstract (2000);Kataoka, S. IBC's 13^(th) Antibody Engineering Meeting. Abstract (2002))harboring human chromosome fragments encoding the human immunoglobulinregion were obtained from Kirin Brewery Co., Ltd., Japan, and werehoused in the animal facility at the La Jolla Institute for Allergy andImmunology. An overview of the technology for producing human antibodiesis described in Lonberg (Lonberg, et al., Int Rev Immunol 13(1):65-93(1995)). Transgenic animals with one or more human immunoglobulin genes(kappa or lambda) that do not express endogenous immunoglobulins aredescribed, for example in, U.S. Pat. No. 5,939,598. Additional methodsfor producing human antibodies and human monoclonal antibodies aredescribed (see, e.g., WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598).Development of bovine carrying human immunoglobulin genes, TC cows, isdescribed in (Kuroiwa, et al., Nat Biotechnol 20(9): 889-94 (2002);Kuroiwa, et al., Nat Genet. 36(7): 775-80 (2004)).

Immunization:

hOX40:hFc recombinant protein was mixed with an equal volume of completeFreund's adjuvant (CFA, Sigma) and an emulsion was prepared. Mice wereimmunized with 10 to 25 μg of protein intraperitoneally and were boostedintraperitoneally with 5 to 10 μg of protein emulsified in incompleteFreund's adjuvant (IFA, Sigma) at two week intervals for 2 to 4 boosts.A final intraperitoneal injection of 5 to 10 μg of soluble hOX40:hFcwithout adjuvant was given five days prior to fusion. Another group ofmice was immunized with hOX40:hFc that was heat denatured by incubationat 80° C. for ten minutes in PBS then mixed 1:1 with RIBI adjuvant(Sigma). Mice were immunized as described above. A third group of micewas immunized with hOX40:hFc conjugated to ovalbumin. The mice wereeither primed with 30 μg of hOX40:hFc-OVA alone or in a mixture withhOX40:hFc, 10 μg and 20 μg respectively, in RIBI. The former received aboost of 30 μg hOX40:hFc-OVA in RIBI, followed by 10 μg of hOX40:hFc inRIBI at two week intervals. A final boost of 20 μg hOX40:hFc-OVA in PBSwas given five weeks later. The latter group's two boosts consisted of 5μg hOX40:hFc-OVA+10 μg hOX40:hFc in RIBI at two week intervals, with afinal boost of 10 μg hOX40:hFc in PBS. All injections wereintraperitoneal. A final group of mice was immunized with CD4+ human Tcells that had been stimulated for two days with PHA (1 μg/ml) andrecombinant human interleukin 2 (IL2, 10 ng/ml, BD Pharmingen, SanDiego, Calif.). Prior to injection the cells received 25 Gy ofirradiation from a cesium source and were diluted 1:1 in RIBI adjuvant.

Hybridoma Production:

The mice with the highest anti-OX40 IgG specific antibody titer in theirserum were selected for the production of monoclonal antibodies. Thespleens were harvested and single cell suspensions were fused to amyeloma cell line (SP2/O—Ag14) (ATCC, Rockville, Md.) at a ratio of 3:1with 50% polyethylene glycol (Boehringer Mannheim, Indianapolis, Ind.).The fusions were plated into 96 well flat bottom plates at an optimaldensity and cultured in complete DMEM-10 medium (Dulbecco's ModifiedEngle's Medium with 10% fetal bovine serum (FBS, Invitrogen Corp.)), 1%non-essential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, 100μg/ml streptomycin sulfate (all from BioWhittaker, Walkersville, Md.),HAT supplement (Sigma), and 10% Hybridoma Cloning Factor (HCF, Biovaris,San Diego, Calif.) in a 10% CO₂, 37° C. incubator. Approximately 4000wells from four fusions were screened by ELISA for human kappacontaining OX40 specific antibodies. Human anti-human OX40 IgGantibodies were confirmed by flow cytometric analysis. Positive wellswere expanded and subjected to three to four rounds of limiting dilutioncloning to obtain monoclonal antibodies.

Antibody and Protein Purification:

For antibody purification, hybridomas were cultured in 2 liter rollerbottles at 350 milliliter to 1 liter/bottle or in a 1 liter Integrasystem (INTEGRA Bioscience, Inc. Ijamsville, Md.) with hybridoma-SFMmedium (Invitrogen Corp.) supplemented with ultra low IgG fetal bovineserum (Invitrogen Corp.). Human OX40:hFc recombinant protein wasgenerated by infecting 1 liter of Tn5 cells for four days. Humanmonoclonal antibodies and OX40:hFc were purified from culture mediausing recombinant Protein A-Sepharose Fast Flow gel (AmershamBiosciences). Conditioned medium generated in roller bottles was firstconcentrated using an Ultrasette tangential flow system (Pall Corp.,East Hills, N.Y.). The conditioned medium was filtered with a 0.22 μmvacuum filter unit (Millipore, Bedford, Mass.) and loaded onto a ProteinA-Sepharose Fast Flow column (Amersham Biosciences) of appropriate sizefor the amount of human antibody in the medium. The column was washedthoroughly with 20 column volumes of PBS and the antibody was elutedwith 0.1 M Gly-HCl, pH 3.6, 0.15 M NaCl and neutralized with 1 MTris-HCl, pH 8.0. The fractions were analyzed by SDS-PAGE and thepositive fractions were pooled and concentrated with a centrifugalconcentrator (Vivaspin, 50,000 MWCO: Sartorius, Gettingen, Germany).Sephadex G-25 desalting columns, (NAP, Amersham Biosciences), were usedfor buffer exchange to PBS, pH 7.4. Finally, the antibody was filtersterilized using syringe filters with 0.22 μm pore diameters and theantibody concentration was determined by the Lowry method. Pyrogencontent was determined using a Limulus Amebocyte Lysate (“LAL”) assay(Associates of Cape Cod, Falmouth, Mass.). The limits of detection ofthis assay are 0.06 EU/mg. If the test was negative, the samples wereconsidered endotoxin free.

Human IgG Quantitation ELISA:

To determine the amount of human antibody present in supernatants andpurified stocks the following protocol was used. Goat anti-human Fcγspecific antibody (Jackson Immunoresearch Laboratories, West Grove, Pa.)was coated to the 96 well plates (Nunc, Denmark) in carbonate buffer at0.5 μg/well for one hour at 37° C. The plates were then blocked withSuperblock (Pierce, Rockford, Ill.) for 30 minutes followed by additionof the samples to the plates. Standard curves were generated using totalhuman IgG (Sigma) or purified human IgG1 or IgG4 (Kirin Brewery Co.,Ltd). The plates were incubated for one hour at 37° C., washed in PBS/1%BSA/0.1% Tween20 (Sigma), and the bound antibody was detected with goatanti-human Fcγ specific antibody conjugated to horseradish peroxidase(HRP, Jackson Immunoresearch) for one hour at 37° C. The TMB substrate(Sigma) was added for 10 minutes and the reaction was stopped with H₂SO₄(LabChem. Inc., Pittsburgh, Pa.). The optical density (OD) was measuredat 450 nm on a microplate reader.

OX40 Specific Antibody Detection ELISA:

Antibody titers, specificity, and production by hybridomas weredetermined by ELISA. In brief, 96 well flat bottom plates were coatedwith 50 μl of hOX40:hFc at 5 μg/ml in carbonate buffer (pH 9.4)overnight at 4° C. or at 37° C. for one hour. After washing twice withPBS/0.1% Tween 20, plates were blocked with PBS/1% BSA/0.1% Tween20 at37° C. for one hour. The serum, supernatant, or purified antibody wasdiluted in blocking buffer, added to the wells, and the plates wereincubated for one hour at 37° C. The plates were washed four times withPBS/0.1% Tween 20 and the peroxidase conjugated sheep anti-human kappadetection antibody (The Binding Site, Birmigham, UK) was added at adilution of 1:2000. Following one hour incubation at 37° C., the plateswere washed and the TMB (Sigma) substrate was added and incubated atroom temperature for ten to thirty minutes. The reaction was stoppedwith H₂SO₄ (LabChem) and the optical density was measured at 450 nm by amicroplate reader.

Flow Cytometry:

Antibody titers, specificity, and relative binding affinities weredetermined by flow cytometric analysis using human OX40 stable CHO-K1transfectants or three day PHA+IL2 activated human PBMC. The cells werewashed once in staining buffer: PBS+2% FBS+0.1% NaN₃+10 mM EDTA, thenresuspended in serum, supernatant, or purified antibodies in a volume of50 μl. The cells were incubated with the antibodies on ice for twentyminutes, washed twice in staining buffer, then resuspended in ananti-human IgG-phycoerythrin labeled secondary antibody. Two differentantibodies were used: (1) goat anti-human IgG (Southern BiotechnologyAssociates, Birmingham, Ala.) or (2) mouse anti-human IgG (BDPharmingen, San Diego, Calif.). Following twenty minute incubation onice, the cells were washed once and fixed ten minutes with 1%paraformaldehyde. After a final wash the cells were resuspended instaining buffer and the samples were acquired using FACScan or FACSCalibur flow cytometers (Becton Dickinson Biosciences, Palo Alto,Calif.), and the data were analyzed using Cellquest (Becton DickinsonBiosciences) or Flow Jo (TreeStar, Inc., San Carlos, Calif.) software.The activated T cells were also stained with mouse anti-human OX40antibody L106 (Becton Dickinson), which was directly conjugated tophycoerythrin or whose binding was detected with anti-mouse IgG-PE(Southern Biotechnology Associates.)

OX40L Blocking Assays:

To determine if the anti-human OX40 antibodies could block OX40L bindingto soluble OX40 both ELISA and flow cytometric protocols were used. Inthe ELISA 96 well Nunc flat bottom plates were coated with recombinantsoluble hOX40:hFc at 2 μg/ml in carbonate buffer (pH9.4) for one hour at37° C. The plates were washed with PBS/0.1% Tween 20 and Superblock(Pierce) was added to the wells to block non-specific binding. The testantibodies were diluted to 1 μg/ml in PBS/Tween and added to the plates.After one hour incubation at 37° C., FLAG tagged recombinant solublehuman OX40L (Alexis Biochemicals, San Diego, Calif.) was added to theappropriate wells without washing out the anti-OX40 antibodies.Following one hour incubation, the plates were washed and ananti-FLAG-peroxidase conjugated antibody (Sigma) was added to the wellsfor one hour at 37° C. The TMB substrate was added, after ten minutesthe reaction was stopped with H₂SO₄, and the results were read at 450 nmusing a microplate reader. In the flow cytometric assay, human CD4+ Tcells were purified and activated with PHA+IL2 for two days. The cellswere washed and resuspended in staining buffer and then incubated withincreasing amounts of human anti-human OX40 antibodies for twentyminutes on ice, from 0.005-50 μg/ml. Soluble recombinant OX40L-FLAG wasthen added to the cells. The cells were washed and incubated withanti-FLAG conjugated to PE (Sigma). After another wash, the cells werefixed with 1% paraformaldehyde and analyzed on a FACScan or FACScalibur.The percent inhibition was determined using the OD or percent positivein the following formula: % inhibition=100−((sample/Maximumbinding)*100).

Anti-OX40 Antibody Cross-blocking Assays:

In order to determine if the antibodies bind the same “epitope” of humanOX40 an ELISA protocol was used. Nunc 96 well flat bottom ELISA plateswere coated with the human anti-human OX40 antibodies in carbonatebuffer at 2 μg/ml for one hour at 37° C. The plates were washed and thenblocked with PBS/1% BSA/Tween 20. Two to 20 μg/ml of the humananti-human OX40 antibodies and the mouse anti human-OX40 antibodies L106(BD Biosciences, San Jose, Calif.), clone 315 (Nichirei Biosciences,Tokyo, Japan), or ACT35 (BD Pharmingen) were then pre-incubated with 2μg/ml recombinant human OX40:mFc fusion protein (Alexis Corporation, SanDiego, Calif.) for thirty minutes at room temperature. The combinationsof antibody-OX40:mFc protein were added to the plate and incubated forone hour at 37° C. After three washes, bound OX40:mFc was detected withperoxidase conjugated sheep anti-mouse Ig (Amersham Biosciences). TheELISA was completed as described above. The percent inhibition wasdetermined using the OD of each sample in the following formula: %inhibition=100−((sample/Maximum binding)*100). To determine if thesehuman anti-human OX40 antibodies block the mouse anti-human OX40antibodies a variation of this ELISA was performed. The method was thesame except the mouse anti-human OX40 antibodies were coated on theplates and 112V8, 112F32, 112Y131, and 112Z5 were pre-incubated withhuman OX40:hFc protein. Binding of the OX40 protein to the coatedantibodies was detected with a sheep anti-human IgG-horseradishperoxidase secondary antibody (Amersham Bisociences).

A flow cytometric assay was also used to determine if the antibodiescrossblock one another. Human CD4 T cells were purified from peripheralblood mononuclear cells as described below and activated with 1 g/mlphytohemaglutinin (Remel, Lenexa, Kans.) and 10 ng/ml recombinant humaninterleukin 2 (BD Pharmingen) for two to three days. The cells werewashed and labeled with 10 μg/ml of the test antibodies for thirtyminutes on ice in PBS supplemented with 2% fetal calf serum, and 0.1%sodium azide. Without washing, biotinylated versions of the sameantibodies were added to the wells at 10 μg/ml and the cells wereincubated for another thirty minutes on ice. The cells were then washedby addition of buffer and spinning at 1200 RPM for three minutes at 4°C. Binding of the biotinylated antibodies was detected by incubationwith streptavidin-phycoerythrin (SA-PE, BD Pharmingen) for twentyminutes followed by another wash as described. The cells were fixed tenminutes in 1% parafomaldehyde. After a final wash the cells wereresuspended in staining buffer and the samples were acquired using aFACS Calibur flow cytometer (Becton Dickinson Biosciences, Palo Alto,Calif.). The data were analyzed using Cellquest (Becton DickinsonBiosciences) or Flow Jo (TreeStar, Inc., San Carlos, Calif.) software.The antibodies tested included 112F32, 112V8, 112Y55, 112Y131, 112Z5,anti-DNP (human IgG1 negative control), mouse anti-human OX40antibodies, clone L106 clone 315, and clone ACT35. The percentinhibition was determined using the following formula: 100−(Geometricmean of test antibody/Max Geometric mean)*100. The antibodies weretested for inhibition of binding of themselves as well as each other.

Purification of Human PBMC from Whole Blood:

Whole blood was collected from healthy donors between the ages of 18 and50 by the normal blood donor program at Scripps Green Hospital (LaJolla, Calif.). Heparin was added to prevent clotting. No race,ethnicity, or gender was specified. The blood was diluted in PBS andthen underlayed with Ficoll-Plaque Plus (Amersham Biosciences). Themononuclear cells were separated from the serum and platelets bycentrifugation at 1800 RPM without the brake. The interface containingthe PBMC was collected and washed two times with PBS.

Purification of CD4+ T Cells:

Human CD4+ T cells were purified using a negative isolation kit fromMiltenyi Biotec. (Auburn, Calif.). The PBMC were labeled with the haptenconjugated antibody mix specific for CD8, CD11b, CD16, CD19, CD36, andCD56 for fifteen minutes at 4° C. in PBS/0.5% BSA/2 mM EDTA. Afterwashing twice the cells were resuspended in the anti-hapten magneticbeads (MACS beads) and the cells were incubated at 4° C. for fifteenminutes. The cells were then washed and applied to a LS/VS+ column thathad been pre-washed and inserted in the magnet. The column was washedthree times with buffer. The “untouched” CD4+ cells were contained inthe flow through from the column Purity was confirmed by flow cytometricanalysis using antibodies specific for human CD3 and human CD4, bothdirectly conjugated to fluorochromes (BD Pharmingen). Alternatively,CD4+ cells were positively selected with CD4 microbeads (MiltenyiBiotec.) The procedure was similar to that described above except thatthe cells that adhered to the column were eluted by removing the columnfrom the magnet and forcing 5 ml of buffer through the column with aplunger.

Mixed Lymphocyte Reaction Assay:

PBMC from two donors were purified and 1×10⁵ cells from each donor wereadded to a 96 well U bottom plate in the presence or absence of humananti-human OX40 antibodies or negative control human IgG4 (anti-humanserum albumin, Kirin Brewery Co., Ltd.) (Ukyo, et al., Immunology109(2):226 (2003)). Antibodies were tested from 0.005 to 100 μg/ml,depending on the study. Multiple donor pairs were tested. The media usedwas RPMI-1640 supplemented with 10% human AB serum (MP Biomedical,Irvine, Calif.), penicillin/streptomycin, L-glutamine, and2-mercaptoethanol. At day six 1 μCi tritiated thymidine (³HTdR) wasadded to each well for the last eighteen hours of culture. The cellswere lysed and transferred to glass filter mats, which were countedusing a scintillation counter (Wallac, Turku, Finland).

Acute Graft Versus Host Disease In Vivo Model:

Severe combined immunodeficient (SCID) male mice aged five to ten weekswere injected with 20 μg rat anti-mouse IL2 receptor β chain antibody(TM131, Tanaka, et al., J Exp Med 178(3): 1103-7 (1993)) to depleteendogenous natural killer cells. The next day the mice received 2.5 Gyof irradiation using a cesium source. Four hours later the mice received10 million total human PBMC in PBS by intraperitoneal injection followedimmediately by intravenous injection of human anti-human OX40 ornegative control hIgG1 (anti-di-nitro-phenol (anti-DNP), Kirin BreweryCo. Ltd.) antibodies at 2, 20, 100, or 200 μg in 100 μl PBS.Alternatively, antibody treatment was delayed until day three or six totest the therapeutic potential of the anti-OX40 antibodies. Mice wereweighed every three to four days and received the anti-IL2Rβ antibodyweekly. At day twelve the mice were killed and assessed for grosspathology. Spleens were removed for flow cytometric analysis, livers andintestines for histology, and serum was collected for human cytokine andantibody analysis (Watanabe, et al., Clin. Immunol. 120:247 (2006)).Chronic Graft Versus Host Disease In Vivo Model: A model of chronic GVHDwas adapted from the acute xenogenic GVHD model (Watanabe, et al., Clin.Immunol. 120:247 (2006)). Disease was induced by the transfer ofpositively selected human CD4 T cells into SCID mice that were preparedin the same was as described above. Mice received one million positivelyselected CD4 T cells by intraperitoneal injection at day 0 and 2, 20, or100 μg anti-OX40 or control antibodies by intravenous injection weeklystarting at day 0. Disease was evident by day thirty and mice wereassessed at day forty-eight. Spleen and lymph nodes were removed forflow cytometry, skin and lung for histology, and serum for humancytokine analysis.

Human Cytokine Analysis:

A panel of eight human cytokines in mouse serum was measured usingmultiplex technology and following the manufacturer's instructions(Bio-Rad Laboratories, Hercules, Calif.).

Purification of Human Natural Killer Cells:

Human natural killer (“NK”) cells were purified using a negativeisolation kit from Miltenyi Biotec. (Auburn, Calif.). The PBMC werelabeled with the biotin conjugated antibody mix specific for T cells, Bcells, stem cells, dendritic cells, monocytes, granulocytes, anderythroid cells for fifteen minutes at 4° C. in PBS/0.5% BSA/2 mM EDTA.This was followed by the addition of anti-biotin magnetic beads (“MACSbeads”). The cells were incubated at 4° C. for fifteen minutes thenwashed and applied to a LS/VS+ column that had been pre-washed andinserted in the magnet. The column was washed three times with buffer.The “untouched” CD56+ NK cells were contained in the flow through fromthe column. Purity was confirmed by flow cytometric analysis usingantibodies specific for human CD56 and human CD3, both directlyconjugated to fluorochromes (BD Pharmingen.)

Antibody Dependent Cell Cytotoxicity Assay (ADCC):

NK cells were purified from human PBMC and cultured for forty-eighthours with 20 ng/ml recombinant human interleukin 2 (BD Pharmingen) inRPMI-1640 supplemented with 10% human AB serum (MP Biomedical, Irvine,Calif.), penicillin/streptomycin, L-glutamine, and 2-mercaptoethanol.These cells were used as effector cells in the Cytotox 96non-radioactive cytotoxic assay (Promega Corp., Madison, Wis.). Thetarget cells were parental or human OX40 transfected EL-4 cells. Fivethousand target cells were incubated with 0.005-10 μg/ml of theanti-human OX40 antibodies or control antibodies for thirty minutes onice in a round bottom 96 well tissue culture plate. Twenty times as manyeffector NK cells were added to the wells in a final volume of 100 μland the plates were spun at 200×g for three minutes before incubation at37° C. with 5% CO₂ for four hours. The plates were spun at 250×g forfive minutes and 50 μl of the supernatants were transferred to an ELISAplate. The assay buffer was added to the plates in a volume of 50 μl.Following a thirty minute incubation at room temperature, 50 μl of thestop solution was added to the wells and the absorbance at 490 nm wasdetermined. The controls required included target cells alone(spontaneous), target cells alone treated with lysis buffer for the lastforty-five minutes of the incubation (maximum), effector cells alone,wells containing media alone with (volume correction) and without theaddition of the lysis buffer. The percent specific lysis is determinedusing the following formula, after the media background is subtractedfrom all wells, and the volume correction value is subtracted from thetarget maximum value. Percent Cytotoxicity=((experimental−effectorspontaneous−target spontaneous)/(target maximum−targetspontaneous))*100.

Isolation of Human Anti-OX40 Antibody Genes:

Cultured hybridoma cells (112V8 (ATCC No. PTA-7219, deposited Nov. 17,2005)), which produce 112V8 antibody (IgG4), were collected bycentrifugation. Total RNA was purified from these cells using RNeasy kit(QIAGEN Inc., Valencia, Calif.) following the manufacturer'sinstructions. SMART RACE cDNA Amplification Kit (Clontech Co., Ltd.,Palo Alto, Calif.) was used for cloning of cDNA that encodes thevariable region of the immunoglobulin genes from total hybridoma cellRNA. Briefly, first strand cDNA was prepared by reverse transcriptasefrom two micrograms of RNA. This cDNA was used as a template forpolymerase chain reaction (“PCR”) to amplify the variable region and apart of the constant region of heavy and light chains (“HV” and “LV,”respectively). The amplified sequences also contained the leadersequences. The reaction was as follows: 2.5 U Pfu Ultra DNA polymerase(Stratagene, La Jolla, Calif.); 0.2 μM 3′ Primer (for Heavy chain:IgG1p, for Light chain: hk5, Table 1); 1× Universal Primer Mix A for the5′ end (UMP primer Mix A included in the SMART RACE Kit); 200 μM dNTPmix; 1 mM MgCl₂; Pfu Ultra Buffer (final concentration is 1×); and cDNAtemplate.

The thermocycling program was five cycles of: 94° C.×30 seconds, 72°C.×3 minutes. Five cycles of: 94° C.×30 seconds, 70° C.×30 seconds, 72°C.×3 minutes. Twenty-five cycles of: 94° C.×30 seconds, 68° C.×30seconds, 72° C.×3 minutes followed by an extension at 72° C.×7 minutes.Amplified DNA fragments were collected by agarose gel electrophoresis,and purified by QIAquick Gel Extraction Kit (Qiagen Co., Ltd., Germany).Purified DNA fragments of HV and LV were integrated into pCR4 Blunt-TOPOvector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen Corp.), andeach construct plasmid was transformed into E. coli, and then cloned.Nucleotide sequences of each insert (HV and LV) in the constructplasmids were analyzed using specific primers (M13F, M13R, Table 1).Based on the sequence obtained from HV and LV, oligonucleotide primerswere designed to amplify VH (V8H38, V8H39) and VL (V8L42, V8L43). (Table1).

112V8 VH and VL were cloned into the IgG1 expression vector. Briefly,oligonucleotide primers, containing 5′-SalI and 3′-NheI restrictionenzyme recognition sites were designed to amplify the variable region ofthe HV by PCR. PCR was performed using pTopoV8VH miniprep DNA as atemplate, V8H38 and V8H39 as primers (Table 1) with Pfu Ultra DNApolymerase. After digestion of the PCR product with NheI and SalI, a 410bp fragment was subcloned into the IgG1 expression vector (IDECPharmaceuticals, San Diego, Calif., N5KG1-Val Lark (a modified vector ofN5KG1, U.S. Pat. No. 6,001,358)) that was pre-digested with NheI andSalI (8.9 kilobases DNA fragment). The existence of variable region ofthe HV was analyzed by restriction digest.

As the second step, LV was inserted into N5KG1-Val Lark-VH vector asfollows: the DNA vector was digested by two DNA restriction enzymes,BglII and BsiWI. The 9.1 kb DNA fragment was isolated. Similarly to theHeavy chain construct, a primer set for PCR of LV was designed tocontain the recognition sites for 5′BglI and 3′BsiWi. These primers,V8L42 and V8L43, were used to amplify VL from the pTopoV8VL miniprepplasmid DNA. The PCR product was digested with BglII and BsiWI andisolated by agarose gel electrophoresis and gel purification. Thisfragment, containing V8VL, was ligated to the prepared 9.1 kb vectorwith T4 DNA ligase and used to transform Top10 cells (Invitrogen Corp.).Positive E. coli transformants were selected. This expression vector,pG1K112V8, was purified, and the presence of both 112V8LV and 112V8HVregions were confirmed by restriction analysis.

112Y55 (ATCC No. PTA-7220, deposited on Nov. 17, 2005), 112Y131 (ATCCNo. PTA-7218, deposited on Nov. 17, 2005), and 112Z5 (ATCC No. PTA-7216,deposited on Nov. 17, 2005) HV and LV variable regions were isolated andsequenced using the same protocol. The primers used for amplification ofthe specific HV and LV are listed in Table 1.

The subclass of an antibody in part determines secondary effectorfunctions, such as complement activation or Fc receptor (FcR) bindingand antibody dependent cell cytotoxicity (ADCC) (Huber, et al., Nature229(5284): 419-20 (1971); Brunhouse, et al., Mol Immunol 16(11): 907-17(1979)). In identifying the optimal type of antibody for a particularapplication, the effector functions of the antibodies can be taken intoaccount. For example, hIgG1 antibodies have a relatively long half life,are very effective at fixing complement, and they bind to both FcRI andFcRII. In contrast, human IgG4 antibodies have a shorter half life, donot fix complement and have a lower affinity for the FcR. Replacement ofserine 228 with a proline (S228P) in the Fc region of IgG4 reducesheterogeneity observed with hIgG4 and extends the serum half life(Kabat, et al., Sequences of proteins of immunological interest 5^(th)Edition (1991).; Angal, et al., Mol Immunol 30(1): 105-8 (1993)). Asecond mutation that replaces leucine 235 with a glutamic acid (L235E)eliminates the residual FcR binding and complement binding activities(Alegre, et al., J Immunol 148(11): 3461-8 (1992)). The resultingantibody with both mutations is referred to as IgG4PE. The numbering ofthe hIgG4 amino acids was derived from Kabat (Kabat, et al., Sequencesof proteins of immunological interest 5^(th) Edition (1991)).

A vector expressing recombinant 112V8 IgG4PE was generated by digestingpG1K112V8 with NheI and BglII to release the fragment containing 112V8VHand 112V8VL. This was ligated to the IgG4PE expression vector(pN5KG4PE-Lark, IDEC Pharmaceuticals, U.S. Pat. No. 6,001,358) cut withthe same enzymes. The resulting plasmid, pG4PEK112V8, was confirmed byrestriction digest.

RNA from 112F32 hybridoma (ATCC No. PTA-7217, deposit date Nov. 17,2005) was used to generate vectors to produce recombinant 112F32G1 and112F32G4 antibodies in the same manner, the 3′ primers used foramplification of the heavy and light chain genes in the RACE reactionswere HH-2 and HK-2, respectively. Amplification of the 112F32HV wasperformed using H725′ and M2H3′. The 112F32LV amplification primers wereF32K5′ and K52D3′ (Table 1). The IgG4 expression vector pN5KG4 wasobtained from IDEC Pharmaceuticals (U.S. Pat. No. 6,001,358.) Theresulting vectors, pKLG1/F32K3H and pKLG4/F32K3H, were confirmed byrestriction enzyme digest and sequencing.

Nucleotide sequence of cDNA of 112F32 HV (from initiation codon (ATG) to the end of variable region)  SEQ ID NO: 3ATGGAGTGGG GGCCGTGCTG GGTTTTCCTT GTTGTTATTT TAGAAGGTGT CCAGTGTGGG 60GTGCAGCTGG TGGAGTCTGG GGGAGGCTTG GTACAGCCTG GGGGGTCCCT GAGACTCTCC 120TGTGCAGCCT CTGGATTCAC CTTCAGTAGC TATAGCATGA ACTGGGTCCG CCAGGCTCCA 180GGGAAGGGGC TGGAGTGGGT TTCATACATT AGTAGTAGTA GTAGTACCAT ATACTATGCA 240GACTCTGTGA AGGGCCGATT CACCATCTCC AGAGACAATG CCAAGAACTC ACTGTATCTG 300CAAATGAACA GCCTGAGAGA CGAGGACACG GCTGTGTATT ACTGTGCGAG AGGAGTGTAT 360CACAATGGCT GGTCCTTCTT TGACTACTGG GGCCAGGGAA CCCTACTCAC CGTCTCCTCA 420Nucleotide sequence of cDNA of 112E32 LV (from initiation codon(ATG) to the end of variable region) SEQ ID NO: 4ATGGACATGA GGGTCCTCGC TCAGCTCCTG GGGCTCCTGC TGCTCTGTTT CCCAGGTGCC 60AGATGTGACA TCCAGATGAC CCAGTCCCCA TCCTCACTGT CTGCATCTGT AGGAAACAGA 120GTCACCATTA CTTGTCGGGC GAGTCAGGAT ATTAGCAGCT GGTTAGCCTG GTATCAGCAG 180AAACCAGAGA AAGCCCCTAA GTCCCTGATC TATGCTGCAT CCAGTTTGCA AAGTGGGGTC 240CCATCAAGGT TCAGCGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGCCTG 300CAGCCTGAAG ATTTTGCAAC TTATTACTGC CAACAGTATA ATAGTTACCC CCTCACCTTC 360GGCCAAGGGA CACGACTGGA GATTAAACGA 390Nucleotide sequence of cDNA of 112V8 HV (from initiation codon(ATG) to the end of variable region) SEQ ID NO: 5ATGGACACAC TTTGCTCCAC GCTCCTGCTG CTGACCATCC CTTCATGGGT CTTGTCCCAG 60ATCACCTTGA AGGAGTCTGG TCCTACGCTA GTGAAGCCCA AACAGACCCT CACGCTGACC 120TGCACCTTCT CTGGATTCTC ACTCAGCACT AGTGGAATGG GTGTGGGCTG GATCCGTCAG 180CCCCCAGGAA AGGCCCTGGA GTGGCTTGCA GTCATTTATT GGGATGATCA TCAACTCTAC 240AGTCCATCTC TGAAGAGCAG GCTCACCATC ACCAAGGACA CCTCCAAAAA CCAGGTGGTC 300CTTACAATGA CCAACATGGA CCCTGTGGAC ACAGCCACAT ATTACTGTGC ACACAGACGA 360GGGGCCTTCC AGCACTGGGG CCAGGGCACC CTGGTCACCG TCTCCTCAGC TTCCACCAA 419GGGC 423 Nucleotide sequence of cDNA of 112V8 LV (from initiation codon(ATG) to the end of the variable region) SEQ ID NO: 6ATGGAAACCC CAGCGCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA TACCACCGGA 60GAAATTGTGT TGACGCAGTC TCCAGGCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC 120CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCAGCTACT TAGCCTGGTA CCAGCAGAAA 180CCTGGCCAGG CTCCCAGGCT CCTCATCTAT GGTGCATCCA GCAGGGCCAC TGGCATCCCA 240GACAGGTTCA GTGGCAGTGG GTCTGGGACA GACTTCACTC TCACCATCAG CAGACTGGAG 300CCTGAAGATT TTGCAGTGTA TTACTGTCAG CAGTATGATA GCTCGCTCAC TTTCGGCGGA 360GGGACCAAGG TGGAGATCAA ACGAACT 387Amino acid sequence of cDNA of 112E32 HV (leader sequence (bold)and variable region) SEQ ID NO: 7MEWGPCWVFL VVILEGVQCG VQLVESGGGL VQPGGSLRLS CAASGFTFSS YSMNWVRQAP 60GKGLEWVSYI SSSSSTIYYA DSVKGRFTIS RDNAKNSLYL QMNSLRDEDT AVYYCARGVY 120HNGWSFFDYW GQGTLLTVSS 140Amino acid sequence of cDNA of 112E32 kappa LV (leader sequence(bold) and variable region) SEQ ID NO: 8MDMRVLAQLL GLLLLCFPGA RCDIQMTQSP SSLSASVGNR VTITCRASQD ISSWLAWYQQ 60KPEKAPKSLI YAASSLQSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQYNSYPLTF 120GQGTRLEIKR 130Amino acid sequence of cDNA of 112V8 HV (leader sequence (bold)and variable region) SEQ ID NO: 9MDTLCSTLLL LTIPSWVLSQ ITLKESGPTL VKPKQTLTLT CTFSGFSLST SGMGVGWIRQ 60PPGKALEWLA VIYWDDHQLY SPSLKSRLTI TKDTSKNQVV LTMTNMDPVD TATYYCAHRR 120GAFQHWGQGT LVTVSSASTK G 141Amino acid sequence of cDNA of 112V8 LV (leader sequence (bold)and variable region) SEQ ID NO: 10METPAQLLFL LLLWLPDTTG EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK 60PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYDSSLTFGG 120GTKVEIKRT 129

TABLE 1 Synthesized DNA primers (SEQ ID NOS: 11-37) No Name Sequence 5′to 3′ Length 11 RACEUPS5′ CTAATACGACTCACTATAGGGC 22-mer 12 IgG1pTCTTGTCCACCTTGGTGTTGCTGGGCTTGTG 31-mer 13 HK5AGGCACACAACAGAGGCAGTTCCAGATTTC 30-mer 14 M13F GTAAAACGACGGCCAGTG 18-mer15 M13R CAGGAAACAGCTATGAC 17-mer 16 V8H38GAGAGAGAGAGCTAGCTGAGGAGACGGTGACCAGGGT 37-mer 17 V8H39AGAGAGAGAGGTCGACCACCATGGACACACTTTGCTCCACG 41-mer 18 V8L42AGAGAGAGAGATCTCTCACCATGGAAACCCCAGCGCAGCTTC 42-mer 19 V8L43AGAGAGAGAGCGTACGTTTGATCTCCACCTTGGTCCCTCC 40-mer 20 HH-2GCTGGAGGGCACGGTCACCACGCTG 25-mer 21 HK-2 GTTGAAGCTCTTTGTGACGGGCGAGC26-mer 22 F725′ ACCGTGTCGACTGGATTCCAAGGCATTTCCAC 32-mer 23 M2H3′GGTGCTAGCTGAGGAGACGGTGAC 24-mer 24 F32K5′ AATCAAGATCTGTCAGGACACA 22-mer25 K52D3′ TATCCCGTACGTTTAATCTCCAGTCGTGTC 30-mer 26 Y131HFAGAGAGAGAGGTCGACCACCATGGACACACTTTGCTCCACG 41-mer 27 Y131HRAGAGAGAGA GGCTAGCTG AAGAGA CGGTGA CCATTGT 37-mer 28 Y131LF5AGAGAGAGA GGTCGACCACCATGG AAACCCCAG CGCAGCTT 41-mer 29 Y131LRAGAGAGAGA GCGTACGTTTGA TTT CCA CCTTGGTCCCTTG 40-mer 30 Y55HFAGAGAGAGAGGTCGACCACCATGGACACACTTTGCTCCACG 41-mer 31 Y55HRAGAGAGAGAGGCTAGCTGAAGAGACGGTGACCATTGT 37-mer 32 Y55LFAGAGAGAGAGATCTCTCACCATGGAAACCCCAGCGCAGCTTC 42-mer 33 Y55LRAGAGAGAGAGCGTACGTTTGATTTCCACCTTGGTCCCCTG 40-mer 34 Z5HFAGA GAGAGAGGTCGACCACCATGACCATGATTACGCCAAGC 41-mer 35 Z5HRGAGAGAGAGAGCTAGCTGAGGAGACGGTGACCAGGGT 37-mer 36 Z5LFAGAGAGAGAGATCTCTCACCATGGAAGCCCCAGCTCAGCTTC 42-mer 37 Z5LRAGAGAGAGAGCGTACGTTTAATCTCCAGTCGTGTCCCTTG 40-mer

Nucleotide sequence of cDNA of 112Y55 HV (from initiation codon(ATG) to the end of the variable region) SEQ ID NO: 38ATGGAAACCC TTTGCTCCAC GCTCCTGCTG CTGACCATCC CTTCATGGGT CTTGTCCCAG 60ATCACCTTGA AGGAGTCTGG TCCTACGCTG GTGAAACCCA CACAGACCCT CACGCTGTCC 120TGCACCTTCT CTGGGTTCTC ACTCAGCACT AGTGGAGTGG GTGTGGGCTG GATCCGTCAG 180CCCCCAGGAA AGGCCCTGGA ATGGCTTGCA CTCATTCATT GGGATGATGC TGAGCGCTAC 240AGTCCATCTC TGAAGAGCAG GCTCACCATC ACCAAGGACA CCTCCAAAAA CCAGGTGGTC 300CTTACAATGA CCAACATGGA CCTTGTGGAC ACAGCCACAT ATTACTGTGC ACACACCCGG 360GGGGCTTTTG ATATCTGGGG CCAAGGGACA ATGGTCACCG TCTCTTCA 408Nucleotide sequence of cDNA of 112Y55 LV (from initiation codon(ATG) to the end of the variable region) SEQ ID NO: 39ATGGAAACCC CAGCGCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA TACCACCGGA 60GAAATTGTGT TGACGCAGTC TCCAGGCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCATC 120CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCAGCTTCT TAGCCTGGTA CCAACAGAAA 180CCTGGCCAGG CTCCCAGGCT CCTCATCTAT GGTGCATTTA GCAGGGCCAC TGGCATCCCA 240GACAGGTTCA GTGGCAGTGG GTCTGGGACA GACTTCACTC TCACCATCAG CAGACTGGAG 300CCTGAAGATT TTGCAGTGTA TTACTGTCAG CAGTATGATA GCTCACGGAC GTTCGGCCAG 360GGGACCAAGG TGGAAATCAA A 381Nucleotide sequence of cDNA of 112Y131 HV (from initiation codon (ATG)to the end of the variable region) SEQ ID NO: 40ATGGAAGCCC TTTGCTCCAC GCTCCTGCTG CTGACCATCC CTTCATGGGT CTTGTCCCAG 60ATCACCTTGA AGGAGTCTGG TCCTACGCTG GTGAAACCCA CACAGACCCT CACGCTGACC 120TGCACCTTCT CTGGATTCTC ACTCAGCACT AGTGGAGTGG GTGTGGGCTG GATCCGTCAG 180CCCCCAGGAA AGGCCCTGGA GTGGCTTGCA CTCATTTATT GGGATGATCA TAGCCCCTAC 240AGCCCATCTC TGAAGAGCAG GCTCACCATC ACCAAGGACA CCTCCAAAAA CCAGGTGGTC 300CTTACAATGA CCAACATGGA CCCTGTGGAC ACAGCCACAT ATTACTGTGC ACGCACCCGG 360GGGGCTTTTG ATATCTGGGG CCAAGGGACA ATGGTCACCG TCTCTTCA 408Nucleotide sequence of cDNA of 112Y131 LV (from initiation codon(ATG) to the end of the variable region) SEQ ID NO: 41ATGGAAGCCC CAGCGCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA TACCACCGGA 60GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC 120CTCTCCTGCA GGGCCAGTCA GGGTGTTAGC AGCTACTTAG CCTGGTACCA GCAGAAACCT 180GGCCAGGCTC CCAGGCTCCT CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC 240AGGTTCAGTG GCAGTGGGCC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT 300GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGCATCCGAC GTTCGGCCAA 360GGGACCAAGG TGGAAATCAA ACGAACTGTG GCTGCACCAT C 381Nucleotide sequence of cDNA of 112Z5 LV (from initiation codon(ATG) to the end of the variable region) SEQ ID NO: 42ATGACCATGA TTACGCCAAG CTTGGTACCG AGCTCGGATC CACTAGTAAC GGCCGCCAGT 60GTGCTGGAAT TCGCCCTTCT AATACGACTC ACTATAGGGC AAGCAGTGGT ATCAACGCAG 120AGTACGGGGG GAGGCTTGGT ACAGCCTGGC AGGTCCCTGA GACTCTCCTG TGCAGCCTCT 180GGATTCACCC TTGATGATTA TGGCATGCAC TGGGTCCGGC AAGCTCCAGG GAAGGGCCTG 240GAGTGGGTCT CAGGTATTAG TTGGAATAGT GATAGTATAG GCTATGTGGA CTCTGTGAAG 300GGCCGATTCA CCATCTCCAG AGACAACGCC AAGAACTCCC TGTATCTGCA AATGAACAGT 360CTGAGAGTTG AGGACACGGC CTTGTATTAC TGTGTAAAAG ATATTAGTGG CTGGTACAGC 420TTTGACTACT GGGGCCAGGG AACCCTGGTC ACCGTCTCCT CA 462Nucleotide sequence of cDNA of 112Z5 LV (from initiation codon(ATG) to the end of the variable region) SEQ ID NO: 43ATGGAAGCCC CAGCTCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA TACCACCGGA 60GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC 120CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG CCTGGTACCA ACAGAAACCT 180GGCCAGGCTC CCAGGCTCCT CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC 240AGGTTCAGTG GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT 300GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGCCGATCAC CTTCGGCCAA 360GGGACACGAC TGGAGATTAA A 381Amino acid sequence of cDNA of 112Y55 HV (leader sequence (bold)and variable region) SEQ ID NO: 44MDTLCSTLLL LTIPSWVLSQ ITLKESGPTL VKPTQTLTLS CTFSGFSLST SGVGVGWIRQ 60PPGKALEWLA LIHWDDAERY SPSLKSRLTI TKDTSKNQVV LTMTNMDLVD TATYYCAHTR 120GAFDIWGQGT MVTVSS 136Amino acid sequence of cDNA of 112Y55 LV (leader sequence (bold)and variable region) SEQ ID NO: 45METPAQLLFL LLLWLPDTTG EIVLTQSPGT LSLSPGERAI LSCRASQSVS SSFLAWYQQK 60PGQAPRLLIY GAFSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYDSSRTFGQ 120GTKVEIK 127Amino acid sequence of cDNA of 112Y131 HV (leader sequence (bold)and variable region) SEQ ID NO: 46MDTLCSTLLL LTIPSWVLSQ ITLKESGPTL VKPTQTLTLT CTFSGFSLST SGVGVGWIRQ 60PPGKALEWLA LIYWDDHSPY SPSLKSRLTI TKDTSKNQVV LTMTNMDPVD TATYYCARTR 120GAFDIWGQGT MVTVSS 136Amino acid sequence of cDNA of 112Y131 LV (leader sequence (bold)and variable region) SEQ ID NO: 47MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQGVS SYLAWYQQKP 60GQAPRLLIYD ASNRATGIPA RFSGSGPGTD FTLTISSLEP EDFAVYYCQQ RSNWHPTFGQ 120GTKVEIK 127Amino acid sequence of cDNA of 112Z5 HV (leader sequence (bold)and variable region) SEQ ID NO: 48MTMITPSLVP SSDPLVTAAS VLEFALLIRL TIGQAVVSTQ STGGGLVQPG RSLRLSCAAS 60GFTLDDYGMH WVRQAPGKGL EWVSGISWNS DSIGYVDSVK GRFTISRDNA KNSLYLQMNS 120LRVEDTALYY CVKDISGWYS FDYWGQGTLV TVSS 154Amino acid sequence of cDNA of 112Z5 LV (leader sequence (bold)and variable region) SEQ ID NO: 49MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP 60GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ 120GTRLEIK 127Production of Recombinant Human Anti-OX40 Antibody from CHO Cells:

For the production of recombinant antibody, the individual antibodyvectors containing the anti-OX40 antibody were electroporated into hostcell dhfr-defective strain of Chinese Hamster Ovary cell (CHO cells,ATCC #CRL-9096) and recombinant antibody was isolated from thesupernatant of the transfected cells. Briefly, ten microgram of purifiedDNA expression vector was linearized by a DNA restriction enzyme, NruI,and the DNA was transfected into 3×10⁶ cells of CHO cells using a BioRad electroporator (0.8 kV, 25 uF). The transfected cells were seeded in96-well culture plates, in EX-CELL 325 PF CHO serum-free medium withglutamine (JRH Bioscience, Lenexa, Kans.), supplemented withpenicillin/streptomycin (BioWhitaker), HT (Sigma), and Geneticin(Invitrogen Corp.) for selecting CHO cells containing the DNA vector.After the selection of several stable transfectant lines, high human IgGproducers were identified by ELISA, and used for production ofrecombinant antibody.

Example 2

The example describes various characteristics of human monoclonalantibodies to human OX40.

KM Mice™ were immunized with soluble recombinant hOX40:hFc in CFA/IFA,hOX40:hFC+hOX40:hFc-ova conjugate in RIBI, heat denatured hOX40:hFc inRIBI, or irradiated, activated human T cells in RIBI. Several of themice raised anti-human OX40 specific antibodies, with a range in humanIgG OX40 specific titers. Splenocytes from the highest responders werefused with myeloma cells to generate human anti-human OX40 producinghybridomas. The production of anti-OX40 antibodies was determined byboth ELISA and flow cytometry using recombinant soluble hOX40:hFc andCHO-OX40 transfectants, respectively. The positive hybridomas werecloned by limiting dilution to yield monoclonal hybridomas. (Table 2)

TABLE 2 Antigens Used for Antibody Generation Antibody Antigen 112F32hOX40:hFc 112V8 Activated human T cells 112Y55 hOX40:hFc plushOX40:hFc-ovalbumin 112Y131 hOX40:hFc plus hOX40:hFc-ovalbumin 112Z5Heat denatured hOX40:hFc

Several antibodies were further characterized for relative bindingaffinity for human OX40, the ability to block human OX40 ligand bindingin vitro, cross-blocking each other, blockade of proliferation in vitro,the ability to block inflammation in vivo, and the ability to mediateADCC. (Table 3).

112F32, 112V8, 112Y131, 112Y55, and 112Z5 all bound specifically toactivated human T cells, but not to resting human T cells (FIG. 1).Human T cells activated for three days were labeled with humananti-human OX40 antibodies at various concentrations and detected withanti-human IgG-PE. The binding of these human anti-human OX40 antibodieswas saturable. The maximum binding of each antibody was determined bytitrating the amount of antibody needed to label activated human T cells(FIG. 2). A range of relative binding affinities were obtained (KD andBMAX, Table 3). The binding of the purified mouse anti-human OX40antibody L106 was also determined and 112F32, 112V8, 112Y55, 112Y131,and 112Z5 all had higher BMAX than L106 on activated T cells.

TABLE 3 Characteristics of Human Anti-Human OX40 Monoclonal AntibodiesOX40L Blocking Labeling of OX40L by Activated T Blocking FACS cells byon BMAX ELISA T cells ADCC % Original KD Geo IC50 IC50 In vitro In VivoLysis Antibody Subclass Epitope μM Mean (μg/ml)  (μg/ml)  EfficacyEfficacy at 10 μg/ml^(@) 112F32 IgG4 A 0.013 103.5 0.650 ni*+{circumflex over ( )} IgG1 +{circumflex over ( )} IgG1 37% IgG4 + IgG4NT 112V8 IgG4 B 0.036 180.8 0.051 0.62 + IgG1 + IgG1 55.6%   IgG4 + IgG418% 112Y55 IgG1 B 0.014 161.8 0.073 0.90 + + 54.2%   112Y131 IgG4 B0.025 170.7 0.069 0.91 + + 18% 112Z5 IgG1 C 0.011 118.2 0.081 14.65 + +35% L106 Mouse L 0.001 52.0 0.982 60.91 NT NT NT IgG1 *ni; notinhibitory at 50 μg/ml {circumflex over ( )}+inhibited proliferation invitro or disease in vivo, not quantitative #: NT; not tested ^(@)Percentlysis with irrelevant antibody at 10 μg/ml was 14%

The OX40 antibodies were analyzed by ELISA to determine if the competewith one another for binding to soluble OX40. The individual humanantibodies were coated in the wells of a 96 well plate. hOX40:mFc waspre-incubated with soluble anti-OX40 antibodies (blocking antibodies)and then added to the coated wells. Binding of hOX40:mFc to the coatedantibody was detected with anti-mouse IgG-HRP. Percent inhibition wasdetermined using the following formula (100−(OD sample/OD maximumbinding sample))*100 (FIG. 3A).

Additionally, mouse antibodies were coated in the wells of a 96 wellplate (L106, 315, ACT35 or mouse IgG) and hOX40:hFc was pre-incubatedwith the blocking antibodies. Binding of the hOX40:hFc was detected withsheep anti-human IgG-HRP (FIG. 3B).

Three “epitope” groups have been identified for these human anti-humanOX40 antibodies. 112V8, 112Y55, and 112Y131 all crossblock one anotherwhile 112F32 and 112Z5 each have unique binding sites. 112V8, 112Y131,and 112Y55 partially reduce binding of 112Z5, but the reverse is not thecase, 112Z5 only blocks itself. This suggests that 112Z5 recognizes adifferent epitope than 112V8, 112Y55, and 112Y131 and any reduction inbinding may be due to steric hinderance. These human anti-OX40antibodies do not block L106, 315 or ACT35 binding to OX40 (FIGS. 3A and3B), while L106 blocks itself and ACT35, it only reduced binding of112V8 and 112Y131 by 10% compared with control antibodies (solid line)that do not bind OX40. This indicates that the antibodies described hererecognize a different epitope than L106. Clone 315 did reduce binding of112V8 and 112Y131, however since these latter antibodies did not block315 binding, they must bind different epitopes.

A flow cytometric assay was also used to evaluate the ability of theantibodies to block binding to surface expressed OX40 on activated humanT cells. Activated T cells were stained with the blocking antibodiesfollowed by addition of biotinylated anti-OX40 antibodies. Additionally,activated human T cells were labeled with mouse anti-human OX40antibodies to block OX40 binding. Binding of the biotinylated anti-OX40antibodies to these cells were detected with SA-PE. The cells wereanalyzed by flow cytometry. The geometric mean of the positive SA-PEstaining was used to calculate percent inhibition using the sameformula, i.e., (100−(OD sample/OD maximum binding sample))*100. Thesedata (FIGS. 4A and 4B) correlate with the ELISA analysis.

Three epitopes are recognized by the human anti-human OX40 antibodies,one by 112F32, a second by 112Z5, and a third by 112V8 and 112Y131. Aswas the case with the ELISA data, 112Z5 only blocked itself, but itsbinding was reduced by 112V8 and 112Y131. The same is true for L106blockade of 112V8 and 112Y131. If L106 is bound to the cells first,112V8 and 112Y131 are still able to bind efficiently to human OX40.However, 112V8 and 112Y131 inhibited L106 binding. These data takentogether suggest that the antibodies sterically hinder L106 frombinding, but do not recognize the same epitope. 112F32 and 112Z5 do notblock L106 binding. ACT35 did not inhibit binding of 112F32, 112V8,112Y131, or 112Z5, but clone 315 did reduce binding of 112V8 and 112Y131by 50%. The binding and detection of antibodies to live cells may induceinternalization of the surface molecule so the level of binding of anindividual antibody may be reduced due to overall lower surfaceexpression of OX40 and not to blocking the binding of the antibodies.Higher affinity antibodies may cause more internalization than loweraffinity antibodies. This may explain the differences between the ELISAand flow cytometric data as well as the differences in results dependingon which antibody is used for blocking.

The ability of 112F32, 112V8, 112Y131, 112Y55, and 112Z5 to blocksoluble hOX40L binding to coated hOX40:hFc fusion protein was testedusing an ELISA protocol (FIG. 5A, Table 3). The plates were coated withhOX40:hFc and after blocking non-specific sites the anti-OX40 antibodieswere allowed to bind. Without washing OX40L-FLAG was added to the wells.Bound ligand was detected with anti-FLAG-HRP secondary antibody. Percentinhibition was determined using the following formula (100−(OD sample/ODmaximum OX40L binding))*100. 112V8, 112Y55, and 112Y131 prevented 85% ofthe binding of soluble OX40L to hOX40:hFc. 112F32 preventedapproximately 70% of the binding of soluble hOX40L to hOX40:hFc and112Z5 blocked 67% of the maximal binding. These data suggest that thesehuman anti-human OX40 antibodies bind to the portion of OX40 that isinvolved in ligand binding.

Slightly different results were obtained when a flow cytometric basedassay was used to monitor blockade of soluble OX40L binding to activatedhuman T cells that express OX40 (FIG. 5B). Activated human T cells werelabeled with anti-OX40 antibodies followed by soluble flag tagged-humanOX40L. Binding of OX40L was detected with anti-FLAG-PE antibody. Percentinhibition was determined using the same formula, i.e.,(100−(sample/maximum binding))*100. In this study, 112F32 was unable toprevent OX40L binding and blockade by 112Z5 was reduced, while the otherantibodies, 112V8, 112Y55, and 112Y131 continued to have similarblocking abilities. The difference in the results may be dependent onthe affinity of 112F32 and 112Z5 for OX40. It may also be due totrimerization of the OX40 protein on the cell surface as opposed todimerization of the soluble OX40:mFc fusion protein.

Alternatively, OX40 expressed on the surface of activated T cells may bein a complex with 4-1BB (Ma, et al., Blood 106(6): 2002-10 (2005)) or aninhibitory molecule similar to BTLA (Cheung, et al., Proc Natl Acad SciUSA 102(37): 13218-23 (2005); Compaan, et al., J Biol Chem280(47):39533-6 (2005); Croft, Trends Immunol 26(6): 292-4 (2005);Gonzalez, et al., Proc Natl Acad Sci USA 102(4): 1116-21 (2005); Sedy,et al., Nat Immunol 6(1): 90-8 (2005)) that prevents 112F32, or reduces112Z5 binding but does not block OX40L binding to OX40. The last threepossibilities may point to the specific epitope bound by the individualantibodies. The epitope recognized by L106, a mouse anti-human OX40antibody, is also dependent on the confirmation of OX40 ligand and OX40,as L106 blocked 95% of OX40L binding to the recombinant OX40 protein,but only 60% of binding to activated T cells. These data indicate thatthere are multiple epitopes on OX40, some of which completely interferewith OX40L binding, while others only partially block the interaction.Binding of these antibodies to the different epitopes may or may nothave unique functional consequences.

Blockade of ligand binding by either method does not necessarily dictatethat OX40 signaling will be impaired by a given antibody. To determineif the human anti-human OX40 antibodies can prevent signaling via OX40,two way mixed lymphocyte cultures were set up using total PBMC fromallogeneic donors. Peripheral blood mononuclear cells from allogeneicdonors were co-cultured in 96 well U bottom plates for seven days in thepresence or absence of various doses of anti-OX40 antibodies or controlantibody in triplicate. 1×10⁵ cells/donor were added per well. Theplates were pulsed for the last eighteen hours of culture with 1μCi/well ³HTdR. The cells were harvested and counted on a scintillationcounter. Proliferation was measured in the presence or absence ofcontrol or anti-OX40 antibodies (FIG. 6). 112V8 (FIG. 6A), 112Y55 and112Z5 (FIG. 6B), and 112Y55, 112Y131, and 112F32 (FIG. 6C) were all ableto reduce the amount of proliferation induced by allogeneic cells in adose dependent manner whereas an negative control hIgG4, anti-humanserum albumin, had no effect on proliferation (FIG. 6A). Multiplestudies were performed and the magnitude of the responses, maximum CPMincorporated, was dependent on the combination of donor PBMC used foreach assay. In mixed lymphocyte cultures both CD4 and CD8 T cellsrespond to the allogeneic antigens. Reduction in the proliferativeresponse by the human anti-human OX40 antibodies may be due to directinhibition of both CD4 and CD8 T cells.

Example 3

This example describes mapping epitopes of human anti-human OX40antibodies with linear peptides of human OX40.

Three peptides within the extracellular domain of human OX40 (Table 4)were chosen to begin mapping of the epitope(s) recognized by 112F32,112V8, 112Y55, 112Y131, and 112Z5.

TABLE 4 Human OX40 Peptides Antibody Peptide Name Peptide Seq 112F32112V8 112Y55 112Y131 112Z5 112A RPAGPGFYNDVVSSKPC - - - - - 112BRAGTQPLDSYKPGVDC - - - - - 112C LAGKHTLQPASNSSDAIC - - - - -

The peptides were generated by A & A Lab, LLC and conjugated to keyholelimpet hemocyanin (“KLH”) with a maleimide linker following themanufacturer's instructions (Pierce). The peptides were coated at 10μg/ml in carbonate buffer to 96 well Maxisorp ELISA plates (Nunc) at 4°C. overnight. The plate was washed with PBS/0.1% Tween 20 and thenblocked with Casein buffer (Pierce) for three hours at room temperature.Candidate antibodies were added at 10 μg/ml diluted in 10% Casein inPBS/0.1% Tween 20. The remainder of the ELISA was performed as describedfor the OX40 specific antibody detection ELISA.

None of the human anti-human OX40 antibodies detectably reacted withthese human OX40 peptides, whereas serum from mice immunized with thepeptides bound the appropriate peptide. These results indicate that112F32, 112V8, 112Y55, 112Y131, and 112Z5 do not detectably bind tothese short linear epitopes from the human OX40 extracellular sequenceunder the assay conditions used. These data do not preclude thesesequences from being recognized by the human anti-human OX40 antibodiesif they were included in longer peptide sequences.

Example 4

This example describes in vivo functional analysis of human anti-humanOX40 monoclonal antibodies.

An acute xenogenic graft versus host disease (XGVHD) model was used totest the therapeutic potential of the 112V8G1 human anti-human OX40antibody in vivo (Watanabe, et al., Clin Immunol 120:247-259 (2006)). Inthis model human peripheral blood mononuclear cells are transferred intosevere combined immunodeficient (SCID) mice. Prior to transfer the miceare treated with an anti-IL2Rβ chain antibody to deplete endogenousmurine natural killer cells, and then they are sub-lethally irradiatedto allow migration of the human cells to the intestinal tract. The humanT cells expand and induce a graft versus host like disease, resulting inweight loss, hematuria, hydroperitoneum, inflammatory cell infiltratesin the liver and intestinal tract, and eventually death. The disease isprimarily mediated by human T cells as transfer of purified T cellsinduces similar symptoms.

SCID mice were treated as described above plus either 112V8G1 or humanIgG1 isotype control (anti-DNP) recombinant antibodies were administeredat day 0 to test the prophylactic potential of OX40 blockade. The micereceived 100, 20, or 2 μg of 112V8G1 antibody or 100 μg anti-DNP byintravenous injection at day 0. Body weight was determined every threeto four days. At day twelve, the mice were killed and analyzed forsymptoms of disease and the spleens were collected for flow cytometricanalysis. The gross pathology observed at day twelve was scored asfollows, diarrhea (0-1), hemorrhaging in the intestine and peritonealcavity, and peritonitis (each 0-5), with the higher number indicatingmore severe disease. The sum of all disease symptoms was used todetermine the total gross pathology score. The mice that received thecontrol antibody all displayed symptoms of XGVHD, with higher pathologyscores than mice that received 112V8G1. All doses of 112V8G1 testedreduced or prevented these symptoms (FIG. 7A.)

Analyses of the spleens were in agreement with this observation. Singlecell suspensions of the spleens were analyzed by flow cytometry for thepresence of human T cells. Human T cells were present in the spleens ofmice treated with the control antibodies, but the number of human Tcells in 112V8G1 treated animals were significantly lower than thenumber of T cells in the control animals (FIG. 7B.) Furthermore,inflammatory human cytokines (interferon-gamma, tumor necrosis factor,and granulocyte-macrophage colony stimulating factor) in the serum weremeasured using multiplex technology. Inflammatory human cytokines werereduced in 112V8G1 treated animals compared with controls. The mostdramatic affect was on the levels of interferon gamma (FIG. 7C.)Appearance of OX40+ T cells in the peripheral blood of allogeneic bonemarrow transplant patients has been correlated with the onset of chronicGVHD (Gadisseur, et al., Bone Marrow Transplant 23(10): 1013-7 (1999);Kotani, et al., Blood 98(10): 3162-4 (2001); Sanchez, et al., Br JHaematol 126(5): 697-703 (2004)).

In order to evaluate the efficacy of OX40 blockade in the treatment ofchronic GVHD, an animal model system of chronic xenogenic GVHD wasestablished. The model is similar to the acute xenogenic GVHD modelexcept that one million purified CD4+ cells are injected into SCID miceinstead of total PBMC. The dose of irradiation is also reduced to 2.12Gyto reduce the level of intestinal damage. The first evidence of diseaseis hair loss and weight loss, followed by death, which is partially dueto infiltration of T cells into the lungs. Disease symptoms becomeapparent around day thirty. Groups of five mice each were treated with112V8G1 recombinant antibody at 100, 20, or 2 μg weekly for five weeksor with anti-DNP hIgG1 control antibody at 100 μg weekly for five weeks.Surviving mice were analyzed at week seven. Gross pathology scores weredetermined by the extent of hair loss, hemorrhaging, and peritonitis(each 0-5) and diarrhea (0-1).

The primary pathology observed was hair loss, and all three doses of112V8G1 reduced this disease symptom compared with control treated mice(FIG. 8A). This observation agreed with the reduction of human T cellsin the spleens of 112V8G1 treated mice at day 48 post transfer of CD4 Tcells (FIG. 8B). In this model the human T cells migrate to the lymphnodes which are easily detected in control animals, but significantlyreduced or not found in anti-OX40 treated animals (FIG. 8C). Note, nolymph nodes were found in the animals treated with 112V8G1 at 20 or 100μg.

Furthermore, unlike the acute model, OX40 positive cells can be easilydetected in the spleens and lymph nodes of control treated mice and thenumber of OX40+ cells was reduced in 112V8G1 treated animals (FIGS. 8Band 8C). Inflammatory cytokines were detected in the serum of mice thatreceived human CD4 T cells and the amount of cytokines detected werereduced in mice treated with 112V8G1 anti-human OX40 compared withcontrol human IgG1. The cytokines produced in this disease model includebut are not limited to interleukin 2, interleukin, 4, interleukin 6,interleukin 8, interleukin 10, granulocyte-macrophage colony stimulatingfactor, interferon gamma and tumor necrosis factor alpha. All cytokinesexcept for interleukin 6 were significantly reduced by treatment with112V8G1, demonstrating the anti-inflammatory activity of OX40 signalinginhibition by depletion and/or blockade.

112V8G4PE was studied in the acute XGVHD model using a prophylacticregimen. This antibody does not have depleting activity, due to itsinability to fix complement or bind Fc receptors and thereforeprevention of disease would be dependent on blockade of OX40 signalingor down-modulation of OX40. A single injection of 112V8G4PE at 200, 20,or 2 μg at day 0 was given to five mice per group. Control treated micereceived 200 μg of anti-DNP human IgG4 antibody at day 0, 3, or 6. Micewere analyzed at day twelve for gross pathology and the number of humanT cells in the spleen (FIGS. 9A and 9B, Table 5). The gross pathology atday twelve was scored on a scale of 0-3 for diarrhea, peritonealhemorrhaging or ascites, and intestinal hemorrhaging, with 0 being nopathology observed and 3 representing severe disease. Both 200 and 20 μgof 112V8G4PE prevented development of overt pathology, while a dose of 2μg was not sufficient to prevent disease. The amount of gross pathologycorrelated with the number of human T cells in the spleen at day twelve.Compared with control treated animals, 200 or 20 μg of 112V8G4PEsignificantly reduced accumulation of human T cells in the spleen whilea dose of 2 μg was ineffective. The differences observed in thetitrations of 112V8G4PE (FIGS. 9A and 9B) and 112V8G1 (FIG. 7) may beattributed to the lack of depleting activity of the 112V8G4PE antibody.

112V8G4PE administration was delayed until day three or six to test thetherapeutic efficacy of blockade of OX40 signaling. Mice were treatedwith 200 μg of 112V8G4PE or control human IgG4 antibody at either daythree or day six following transfer of human PBMC. Gross pathology wasassessed at day fourteen. Blockade of OX40 signaling ameliorated diseasewhen 112V8G4PE anti-human OX40 antibody was given at either day three orday six compared with the level of disease observed in control antibodytreated animals (FIG. 9C). The number of human T cells in the spleens ofanti-OX40 treated mice was also significantly reduced compared withcontrol treated mice. 112V8G1 also reduced disease if administered atday six. These data demonstrate the efficacy of OX40 blockade by OX40specific antibodies as a therapeutic approach to reduce diseasepathology.

In both the acute and chronic xenogenic models of graft versus hostdisease 112V8, a human anti-human OX40 antibody, significantly reduceddisease pathology, production of inflammatory cytokines, and the numberof human T cells in the spleens and lymph nodes compared with controltreated animals. These data demonstrate that directly targeting OX40positive cells is a suitable therapy for the prevention (prophylactic)and amelioration (therapeutic) of T cell-mediated diseases. Similarresults were obtained in one or both models with 112F32, 112Y55,112Y131, and 112Z5 (Table 3).

In the model of acute GVHD, both CD4 and CD8 human T cells are requiredfor disease. The CD4 T cells are necessary for induction, while the CD8T cells mediate the majority of the pathogenesis. Prevention andamelioration of disease by administration of one of the described humananti-human OX40 antibodies could be due to direct antagonism of both CD4and CD8 T cells, or may be due to direct blockade of CD4 T cells withindirect affect on CD8 T cells by reducing CD4 help. In addition, OX40antibodies may promote generation or increase numbers of regulatory Tcells. Although not wishing to be bound by any theory, it is possiblethat one or all of these mechanisms may mediate amelioration of disease.

Although not wishing to be bound to any theory, a potential mechanism ofaction of these anti-human OX40 antibodies is to induce antibodydependent cell cytotoxicity (ADCC) by natural killer cell or neutrophileffector cells. This process is dependent on the ability of the Fcreceptors expressed by the effector cells to bind to the anti-OX40antibodies bound to OX40 expressed on activated T cells. The ability ofimmunoglobulins to bind Fc receptors is determined by the subclass ofthe antibody and by the type of Fc receptor. Human IgG1 antibodies bindto Fc receptors on natural killer cells and neutrophils while human IgG4antibodies do not (Huber, et al., Nature 229(5284): 419-20 (1971);Brunhouse, et al., Mol Immunol 16(11): 907-17 (1979)). The humananti-human OX40 antibodies 112F32 (IgG1), 112V8 (IgG1 and IgG4PE),112Y55 (IgG1), 112Y131 (IgG4), and 112Z5 (IgG1), were tested for theirability to mediate ADCC of OX40 expressing target cells by human naturalkiller cells.

A non-radioactive cytotoxicity assay was used to determine the percentspecific lysis of the target cells following a four hour incubation withthe human anti-human OX40 antibodies and human natural killer cells at aratio of twenty effector cells to one target cell (FIG. 10). EL4-humanOX40 target cells were labeled with 0.001-10 μg/ml of anti-OX40 ornegative control antibodies then incubated with human natural killercells. Lysis of the target cells was determined by release of lactosedehydrogenase in a non-radioactive cytotoxicity assay. The percentspecific lysis was determined as described in the methods. The humanIgG1 anti-human OX40 antibodies induced ADCC of EL4-human OX40 targetcells in a dose dependent manner. The human IgG4 anti-OX40 antibodiesand a control human IgG1 antibody did not induce specific lysis of thetarget cells. The level of ADCC activity correlates with the relativebinding affinity and the epitope group as those antibodies in epitopegroup B (112V8 and 112Y55) both had higher ADCC activity than antibodiesin groups A (112F32) and C (112Z5).

The results of the analyses described here identify antibodies with arange of binding affinities, three different epitopes, and demonstrationof their efficacy in blocking OX40 ligand binding, and preventing orameliorating T cell mediated inflammatory reactions. The ability ofthese human anti-human OX40 antibodies (112F32, 112V8, 112Y55, 112Y131,and 112Z5) to reduce proliferation, disease progression and inflammatorycytokine production demonstrates the potential of direct blockade ofOX40 as an approach for treatment of T cell mediated inflammatory orautoimmune diseases, including but not limited to rheumatoid arthritis,multiple sclerosis, psoriasis, Crohn's disease, graft versus hostdisease, and transplantation rejection. Depleting activity of theanti-OX40 antibodies is not necessary for reduction in proliferation oramelioration of disease, which may reduce potential adverse effects dueto T cell depletion.

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What is claimed:
 1. An isolated or purified monoclonal antibody, or afragment thereof, that specifically binds to an epitope in an amino acidsequence OX40 extracellular domain, wherein the antibody comprises aminoacid sequences of all CDRs of a heavy chain variable region and a lightchain variable region of an antibody which is produced by a hybridomacell line denoted as 112V8, deposited as ATCC Accession No. PTA-7219. 2.The monoclonal antibody, or the fragment thereof, of claim 1, whereinthe epitope in an amino acid sequence of OX40 extracellular domain iswithin an amino acid sequence set forth as: (SEQ ID NO: 50)MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSNDRCCHECRPGN GMVSRCSRSQ NTVCRPCGPG FYNDVVSSKPCKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYKPGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ GPPARPITVQ PTEAWPRTSQ GPS.


3. The isolated or purified monoclonal antibody, or the fragmentthereof, of claim 1, wherein the antibody comprises the amino acidsequence of VH and VL of the antibody which is produced by a hybridomacell line denoted as 112V8, deposited as ATCC Accession No. PTA-7219. 4.The monoclonal antibody, or the fragment thereof, of claim 1, whereinthe antibody comprises: a heavy chain variable region comprising anamino acid sequence from the amino acid at position 20 of SEQ ID NO:9 tothe amino acid at position 141 of SEQ ID NO:9, and a light chainvariable region comprising an amino acid sequence from the amino acid atposition 21 of SEQ ID NO:10 to the amino acid at position 129 of SEQ IDNO:10.
 5. The fragment of the monoclonal antibody of any one of claim1-3 and 4, wherein the fragment is selected from Fab, Fab′, F(ab′)₂, Fv,Fd, single-chain Fvs (scFv), or disulfide-linked Fvs (sdFv).
 6. Themonoclonal antibody of any one of claim 1-3 and 4, wherein the OX40 ishuman OX40.
 7. The monoclonal antibody of any one of claim 1-3 and 4,wherein the OX40 comprises a sequence: (SEQ ID NO: 50)MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI.


8. The monoclonal antibody, or the fragment thereof, of any one of claim1-3 and 4, wherein the antibody is an IgG1, IgG2, IgG3, IgG4, IgA, IgM,IgE, or IgD isotype.
 9. The monoclonal antibody or, the fragmentthereof, of any one of claim 1-3 and 4, wherein the antibody orsubsequence further comprises a heterologous domain.
 10. Apharmaceutical composition comprising the monoclonal antibody or thefragment thereof of any one of claim 1-3 and 4, and a pharmaceuticallyacceptable carrier or excipient.
 11. The monoclonal antibody, or thefragment thereof, any one of claims 1 and 2, wherein the antibodycomprises a human, humanized or chimeric antibody.
 12. The monoclonalantibody, or the fragment thereof, any one of claims 1-3 and 4, whereinthe antibody has OX40 antagonist activity.